Title of Invention

"A METHOD FOR PRODUCING AN IN VITRO EXPRESSION LIBRARY"

Abstract A method for producing an in vitro peptide expression library comprising a plurality of peptides, wherein each peptide is linked to the DNA construct encoding the peptide, comprising the steps of: (a) providing a DNA construct comprising: (i) a DNA target sequence; (ii) DNA encoding a library member peptide; and (iii) DNA encoding a peptide capable of non-covalently binding directly or indirectly to said DNA target sequence of (i); wherein said DNA construct and encoded protein are selected to have cis-activity (b) expressing a plurality of DNA constructs according to (a) wherein said DNA constructs encode a plurality of library member peptides such that each expressed peptide is non-covalently linked to the DNA from which it was produced.
Full Text This invention relates to a method for producing an in vitro expression library.
Field of the Invention
The present invention relates generally to recombinant DNA technology and, more particularly, to in vitro methods for constructmg and screening DNA libraries for DNA sequences that encode biologically active molecules.
Background of the Invention
Isolating an unknown gene which encodes a desired peptide from a recombinant DNA library can be a difficult task The use of hybridisation probes may facilitate the process, but their use is generally dependent on knowmg at least a portion of the sequence of the gene which encodes the protein When the sequence is not known, DNA libraries can be expressed m an expression vector, and antibodies have been used to screen plaques or colonies for the desired protein antigen. This procedure has been useful in screening small libraries, but rarely occurring sequences which are represented in less than about 1 in 105 clones, as is the case with rarely occurring cDNA molecules or synthetic peptides, can be easily missed, making screemng libraries larger than 106 clones at best laborious and difficult. Screening larger libraries has required the development of methods designed to address the isolation of rarely occumng sequences, which are based on the co-selection of molecules, along with the DNAs that encode them In vivo methods have been developed to screen large libraries, such as phage display and "peptides on plasmids" using lad fusions of peptides
Phage display is based on DNA libraries fused to the N-termmal end of filamentous bacteriophage coat proteins and then expression m a bacterial host resulting m the display of foreign peptides on the surface of the phage particle with the DNA encoding the fusion protem packaged m the phage particle (Smith G P., 1985, Science 228' 1315-1317) Libraries of fusion proteins incorporated into phage, can then be selected for binding members against targets of interest (ligands). Bound phage can then be allowed to reinfect Escherichia cob (E coli) bacteria and then amplified and the selection repeated, resulting in the ennchment of binding members

(Parmley, S. F., & Smith, G. P. 1988 , Gene 73 305-318, Barrett R W et al. 1992, Analytical Biochemistry 204. 357-364 Williamson ct al, Proc Natl Acad. Sci USA, 90 4141-4145; Marks et al. 1991, J. Mol Biol. 222: 581-597)
Lad fusion plasmid display is based on the DNA binding ability of the lac repressor. Libraries of random peptides are fused to the C-termmal end of the lad repressor protein. Linlcage of the Lacl-peptide fusion to its encoding DNA occurs via the lacO sequences on the plasmid, forming a stable peptide-Lad-peptide complex These complexes are released from their host bacteria by cell lysis, and peptides of interest isolated by affinity purification on an immobilised receptor target. The plasmids thus isolated can then be reintroduced into E. coli by electroporation to amplify the selected population for additional rounds of screening (Cull, M G et al 1992 Proc. Natl Acad Sci U S.A. 89-1865-1869)
These bacterial methods are limited by the size of the library that can be created by current methods of introducing DNA into host bacteria, the potential cellular toxicity of the expressed peptides introduced, and by the inability to introduce post-translational modifications, or to incorporate novel amino acids into the expressed peptide.
An entirely in vitro nbosome system has been described based on the linkage of peptides to the RNA encoding them through the ribosome (WO91/05058). Ribosome display has also been used for the selection of single-chain Fv antibody fiagments (scFv) (Matheakis, L. C. et al, 1994 Proc Natl Acad. Sci. USA, 91: 9022-9026, Hanes, J. & Pluckthun, A 1997 Pioc. Natl. Acad. Sci. USA, 94 4937-4942) This method suffers from the lower stability of the RNA genetic material and the increased degradation likely under certain selection conditions where RNAse is likely to be present.
The in vitro method described by Griffiths and Tawfik (WO 99/02671 and WO 00/40712) addresses some of these concerns by compartmentalizing DNA prior to expression of peptides, which then modify the DNA within the compartment Peptides capable of modifications, resulting from enzymatic activity of interest, are then selected m a subsequent step However, no direct selection of peptide binding
activity is possible of both peptide and DNA without modification of the DNA encoding that peptide, and by leleasmg the modified DNA from the compartment.
Another prior art method, covalent display technology, or CDT, is described in W09837186. This method relies on covalent linkage of protein to DNA to letain the linkage of genotype to phenotype, through the cis action of the crosslinkmg protem. This method teaches that two requirements are needed for successful use of this teclimque. Firstly, proteins are lequired which interact in vitro with the DNA sequence which encodes them (cis action), and secondly, said proteins must establish a covalent linlcage to their own DNA template This method suffers fi-om the fact that the DNA is chemically modified which can prevent the recovery and identification of the binding peptide of interest.
There remains a need for more versatile in vitro methods of constructing peptide libraries in addition to the methods described above, which can allow direct selection of binding activity, as well as for enzymatic activity, and that allow efficient production of complex peptide structures, while still allowing recovery of intact genetic material encoding the peptide of interest.
Summary of the invention
The present invention therefore provides a method for producing an in vitro peptide expression library comprising a plurality of peptides, wherein each peptide is linked to a DNA construct encoding the peptide, comprising the steps of:
(a) providing a DNA construct comprising
(i) a DNA target sequence,
(n) DNA encoding a library member peptide; and
(lii) DNA encoding a peptide capable of non-covalently binding
directly or indirectly to said DNA target sequence of (ii); v/herein said DNA construct and encoded protein are selected to have cis-activity;
(b) expressing a plurality of DNA constructs according to (a), wherein
said DNA constructs encode a plurality of library member peptides
such that each expressed peptide is non-covalently linked to the DNA
from which it was produced Also provided is a method for producing an in vitro peptide expression library comprising a plurality of peptides, wherein each peptide is hnlced to the DNA construct encoding the peptide, comprising the steps of
(a) providing a DNA constiuct comprising
(i) DNA encoding a library membei peptide, and
(ii) DNA encoding a peptide capable of non-covalently binding to
a biflinctional agent, wherein said DNA construct and encoded protein are selected to have cis-activity,
(b) binding a bifunctional agent or a DNA tag capable of binding a bifunctional agent to said DNA construct of (a), wherein said bifunctional agent is capable of binding to the peptide encoded by said DNA of (ii); and
(c) expressing a plurality of DNA constrcuts according to (b), wherein said DNA constructs encode a plurality of library member peptides such that each expressed peptide is linked via said bifunctional agent to the DNA from which it was produced.
The present invention extends to the peptide iibrares produced by such methods and to the DNA constructs used m such methods.
The present invention also provides methods of screening in vitro peptide expression libraries of the inention In one aspect there is provided a method of identifying and/or purifying a peptide exhibiting desired properties from an in vitro peptide expression library produced according to the method of any one of the preceding claims, compriing at least the steps of (a) screening said library and (b) selecting and isolating the relevant library member, In a second spect there is provided a method of identifying a specific ligand binding peptide, said method comprising at least the steps of (a) screening an in vitro peptide exprression library produced according to the method of the invention with hgand molecules which are optionally bound to a solid support; (b) selecting and isolating a library member
binding to said target molecule, and (c) isolating the peptide which binds specifically to said target molecule In a thud aspect there is provided a method of identifying and/or purifying a peptide having the ability to bind a specific DNA target sequence comprising at least the steps of (a) providing an in vitro expression library according to the invention wherein said peptide or protein of (iii) is a library member peptide having DNA binding activity and wherein said DNA target sequence of (i) is the target sequence of interest, (b) selecting and isolating a library member in which the encoded protein binds to said target sequence, (c) isolating the peptide which binds to said target sequence.
In addition to isolating and/or identifying specific peptides from the libraries of the invention, the screening methods of the invention may be used to isolate and/or identify the DNA encoding specific peptides from the library
Brief Description of the Figures
Figure 1 gives a schematic representation of a method by which a DNA construct of the invention may be linked to the peptide that it encodes.
Figure 2 give a schematic representation of a method of the invention by which a DNA binding protein may be converted to a cis-acting DNA binding protein.
Figure 3 gives a schematic representation of how a target sequence specific DNA binding protein may be isolated from a library of the invention.
Figure 4 gives a schematic representation of how a library protein may be linked to its coding DNA through cis action and the use of a bi-specific binding molecule
Figure 5 demonstrates cis activity. 1 1 mixture of two different sized input DNAs (CK-RepA or V5-RepA) selected against either antibody. 1-Marker DNA; 2-PCR amplification after selection on anti-human CK antibody, 3-PCR amplification after selection on anti-V5 peptide antibody
Figure 6 shows the specificty of anti-V5 antibody binding clones ELISA screening, read at 450nM, of the seven clones (1-7) that show specific binding to anti-V5 antibody The bars m group of four represent the ELIS A signal of the clones screened against from left to right; anti-human kappa region antibody, anti-V5
antibody, BSA, and blank A negative control that neither express CK nor V5 is also presented (8).
Figure 7 shows culture supernatant ELISA OD 450mB signals for peptides recovered after 5 rounds of selection against B globigii spores in Example 4. A = clonele; B. = clonelf; C = clonelg, D = cioneSa, E = clonelOc, F. = clonelOe, G = negative control.
Figure S shows OD 450nm signals for peptides isolated after 4 rounds of selection against anti-V5 antibody in Example 5 A. = PI CI 2, B = P2H1; C. = P1B5; D. = P2B8. Peptide-phage were tested against anti-V5 and anti-ACTH peptide antibodies
Figure 9 shows OD 450mn signals for S3aithetic peptides isolated after 4 lounds of selection against ovalbumin. A = CI; B = C4, C = C6, D - C8, E. = negative control Peptides were tested against ovalbumin, anti-V5 antibody and blocked plate (plastic).
Figure 10 shows PCR recoveries of scFv DNA after selection on BSA oi BSA-mecoprop A Anti-mecoprop scFv selected on BSA, 2.5mM ox-glutathione. B. Anti-mecoprop scFv selected on mecoprop-BSA, 2.5mI\'I ox-glutathione. C. Airti-mecoprop scFv selected on BSA, no ox-glutatlaione D Anti-mecoprop scFv selected on mecoprop-BSA, no ox-glutathione
Brief Descrintion of the Sequences
SEQ ID Nos 1 to 11, 19 to 23, 26 to 35 and 45 to 47 show the primers used in the Examples
SEQ ID NO: 12 shows the sequence of the TAC-MYC-CK-REPA-CIS-ORI construct, SEQ ID NO. 13 shows the sequence of the TAC-MYC-V5-REPA-CIS-ORJ construct, SEQ ID NO 24 shows the sequence of the TAC-V5-REPA-CIS-0RI-408 construct and SEQ ID NO 25 shows the sequence of the TAC-NNB-REPA-CIS-ORI-408 construct
SEQ ID NO: 14 shows the estrogen receptor target recognition sequence.
SEQ ID Nos 15 and 16 show the DNA and amino acid sequences of the lepA gene from the Rl plasmid of the incFII incompatibility group SEQ ID Nos 17 and
18 show the sequences of the CIS DNA element and on sequence form the same system.
SEQ ID Nos 36 to 39 show the sequences of peptides isolated after selection in Example 5 SEQ ID Nos 39 to 43 show the sequences of clones isolated in Example 6
Detailed Description of the Invention
The present invention relates to the constiuction and screening of a library for a nucleotide sequence which encodes a peptide of interest in vitro. The constructs encoding the peptide of interest are designed such that the expressed peptide shows CIS activity for the construct Cis activity is defined as the ability of the peptide to bind to the DNA from which the peptide was produced, i.e from which it was transcribed and translated In vitro expression of the construct results in binding of the peptide to the DNA encoding that same peptide molecule by non-covalent interaction. This differs from the teaching of WO 98/37186, which does not allow for the possibility of in vitio non-covalent interaction between protein and the DNA it encodes, and indeed specifically excludes such interactions from having any practical use for library screening
Non-covalent binding refers to an association that may be disrupted by methods well known to those skilled in the art, such as the addition of an appropriate solvent, or a change m ionic conditions, for example, the addition of low pH glycine or high pH triethylamine In the present case, a typical example of non-covalent binding would be the non-covalent interaction between a DNA binding protein and a DNA molecule. Conversely, when a covalent linkage is formed between the DNA and the encoded polypeptide, the displayed peptide or protein will not be released from the DNA by ionic conditions and solvents that would disrupt non-covalent DNA binding protein DNA interactions For example, the bacterial leplication protem rep A binds non-covalently to its target DNA sequence oriR and can be released from this target DNA sequence at salt concentrations greater than 0.2M KCl (Giraldo R, &DiazR. 1992 J Mol Biol 228- 787-802) This salt concentration would not affect a covalent linkage, which would require much harsher conditions to
release the covalently bound protein, with the increased lisk of damage to the lecovered DNA
The current invention describes cis activity and non-covalent binding which allow the encoded peptide or protein to remain associated with the DNA construct with a half life sufficient to allow individual peptides and the associated DNA encoding that peptide with an activity of interest to be separated from the resulting mixture of protein DNA complexes For example, the association between the encoded protein and its DNA may have a half life of up to 30 minutes, up to 45 minutes, up to one hour, up to 2 hours, up to 6 hours or up to 12 hours. The screening methods of the invention may therefore be carried out immediately after construction of the library, or later, for example up to one, up to two, up to six, up to twelve hours or up to twenty four hours or more than twenty four hours later.
Surprisingly, therefore, the invention described herein demonstrates that such encoded peptides or proteins can be expressed in vitro and bound to the DNA encoding that peptide m the presence of other DNA sequences The invention also demonstrates that covalent linkage between protein and DNA is not required to maintain such cis activity, and that a non-covalent interaction between DNA and binding protein is sufficient to allow selection of peptides in an in vitro expression and selection system
According to the present invention, individual DNA library members, each of which encodes a peptide to be expressed m the peptide expression library (library member peptide), are placed in a suitable DNA construct The DNA construct into which the DNA library member is placed includes all the sequences necessary to allow expression of the library member peptide from the construct and to allow the peptide encoded by the construct to bind to the DNA construct which encoded it Each peptide in the library will typically comprise a fusion protein comprising the library member peptide fused to a peptide involved in binding of the fusion protein to the relevant DNA construct. Such fusion proteins may comprise further sequences and said library peptide may be joined to said binding peptide via a linker sequence
A plurality of such constructs, encoding a plurality of different library member peptides form a DNA library of the invention Expressing such a library of
DNA molecules results in the non-covalent binding of individual encoded proteins to the DNA which encoded them and from which they have been transcribed and translated, in the presence of many other DNA molecules that encode other members of the library The sequence encoding the peptide library member present m a paiticular- encoded protein will therefore be present m the DNA which is bound to that protem. This process therefore links the library member peptide, in a biologically active form (usually having a binding activity) to the specific library member DNA sequence encoding that peptide, allowing selection of peptides of interest, for example due to a particular binding activity, and subsequent isolation and identification of the DNA encoding that library member peptide.
For the purposes of the invention a DNA library is therefore a population of DNA constructs. Each construct comprises a DNA sequence encoding a peptide to be expressed as a library member peptide and each contains appropriate promoter, translation start and stop signals A DNA library of the invention will contain a plurality of such DNA molecules. A plurality of DNA constructs are provided each encoding a library member peptide to provide a plurality of different library members. Preferably a DNA library will contain at least 104 discrete DNA molecules. For example, a DNA library may contain more than 106, more than 108, more than 108 more than l012' or more than l014' discrete DNA molecules.
A peptide expression library is defined as a population of peptide sequences expressed from a library of DNA molecules. A peptide expression library of the present invention therefore encompasses a library of peptides which are non-covalently bound to the DNA which encoded them. For example, a peptide expression library of the present invention may be a library of at least 104 discrete proteins each comprising a library peptide sequence, expressed from a library of DNA molecules A peptide expression library of the invention may be any library formed by the expression of a DNA library of the present invention
A peptide library member can be defined as an amino acid chain of variable composition of at least two amino acids m length, or part or all of a naturally occurring protein such as an enz3'me, a binding molecule such as a receptor or an antibody or a fragment thereof A suitable peptide library member may be a peptide
having random amino acid composition The peptide of variable or random composition may be flanked by known amino acid sequences a the N- and/or C-terminus to constrain the structure These known sequences may vary in length. The peptide of variable or random composition may be inserted at various positions in a known protein scaffold, such as a receptor or antibody or other protein or fragment thereof The peptide may be inserted into the same protein scaffold once or more than once, for example two or more times
A DNA construct according to the present invention may comprise DNA encoding a library member peptide and means for the encoded peptide to bind to the encoding DNA construct. In addition to DNA encoding a library member peptide, a suitable DNA construct of the invention comprises at least a DNA target sequence and DNA encoding a peptide capable of binding directly or indirectly to the DNA target sequence.
According to the present invention, the DNA constarct and the encoded protein are chosen to have cis-activity. That is, the encoded protein has the ability to bind specifically to the DNA molecule which encoded it. For example, cis-activity may function to allow the encoded DNA binding peptide to bind specifically (directly or indirectly) to the target sequence of the DNA construct which encoded it rather than to the target sequence of another DNA construct
In some cases, cis activity may be provided due to the presence of a DNA element that directs cis-activity, i.e that allows or forces the protem encoded by the DNA construct to have cis-activity, and therefore to bind to its encoding sequence. In other cases, a separate DNA element per- se may not be required where the nature of the encoding DNA inherently confers cis activity on the encoded peptide. A DNA element that directs cis-activity may be provided m the DNA construct together with the DNA encoding a peptide that interacts with that cis element for example, in the case of the cis element from the repA system discussed below, DNA encoding a fragment of the lepA sequence comprising at least 20 ammo acids from the C terminal of repA may be provided along with the cis DNA element. It may be possible to confer cis activity upon a DNA binding peptide that is not normally cis-acting by including in the DNA construct such a DNA element and any
further sequences necessary for its action. for example, DNA encoding a peptide that interacts with the cis element used may be included in the DNA construct
Alternatively, a peptide that interacts with the cis element may be part of the DNA binding peptide. For example, the DNA binding peptide may be repA which comprises the sequence that interacts with the repA cis element Alternatively, the DNA binding peptide may bind to its encoding DNA in cis without the need for a separate cis element
A suitable DNA element may be any element which allows or directs cis-activity Such a DNA element may act. for example, by interacting with the machinery involved in translation and transcription of the DNA construct to delay the production and release of the encoded peptide.
Any DNA element which allows the encoded peptide to bind specifically to the DNA molecule which encoded it may be used as a DNA element according to the present invention One example of a suitable DNA element is that of the repA-cis system described in more detail below. In that system, RNA polymerase is paused by loops in the 5' cis sequence prior to the rho dependent termination of transcription. The action of the DNA element therefore allows the encoded binding peptide to bind to the DNA target sequence in the construct from which it was produced.
Preferably, the cis DNA element will be be located 3' m the DNA construct to the library member peptide and to the peptide or protein capable of binding to the DNA target sequence This means that these sequences may be transcribed and translated before the RNA polymerase reaches the cis acting sequence
According to the present invention, the binding peptide may be linked to the DNA construct directly or indirectly In the case of direct binding, the binding peptide binds directly and non-covalently to the DNA target sequence In the case of indirect binding, the link between the binding peptide and DNA construct is provided by a further molecule Such a molecule, for example a bifunctional agent as described below, will associate with both the peptide and the DNA target sequence
A suitable DNA construct may compuse further sequences, for example suitable promoter sequences to allow expression of the encoded peptide.
One example of a system in which cis-activity exists is the a cis acting incompatibility group plasmid replication protem, termed rep A, system. Aspects of this system may be utilised m the present invention as explained below
Numerous plasmids include sequences encoding lepA and cis DNA elements The rep A sequence and cis DNA element present m a DNA construct of the invention may be derived from the same plasmid strain or may be derived from different plasmid strains
It is believed that the repA-cis system acts as shown m Figure 1. Briefly, RNA polymerase is paused by loops in the 5'-CIS sequence prior to rho dependent termination of transcription This allows transient C-terminal repA peptide interaction with CIS, and possibly DNA bending RepA peptide then binds to ori, which is a defined distance away from the teraminal amino acid of the repA coding sequence (Prazkier et al 2000 J Bacteriology 182 3972-3980, Praszkier and Pittard 1999 J Bactenol 181. 2765-2772, Masai and Arar 1988 Nucleic Acids Res. 16: 6493-6514)
The compatibility of a RepA sequence from a plasmid with a cis sequence from another plasmid can be readily determined by monitoring for the interaction of RepA with the selected cis sequence
Suitable repA proteins and sequences and cis DNA elements include those of the IncI complex plasmids or the IncF, IncB, IncK, LncZ and IncL/M plasmids, which are distantly related at the DNA level, but which control plasmid replication tlirough the action of the cis acting repA protein (Nikoletti et al. 1986 J Bacteriol 170:1311-1318; Prazkier J etal. 1991J Bactenol 173.2393-2397). Specific plasmids which may be used to provide these sequences include the Rl plasmid of the IncII incompatibility group and the incB plasmid pMU720 (described by Praskier J. & Pittard J. 1999 Role of CIS in lephcation of an IncB plasmid J Bacteriol. 181: 2765-2772). The DNA and amino acid sequences of repA derived from the Rl plasmid of IncII are given m SEQ ID Nos. 15 and 16 The DNA sequence of the cis DNA element from the Rl plasmid of IncII is given in SEQ ID NO- 17. Typically, the cis element is 150 to 200 nucleotides in length. Shorter or larger sequences may be used, so long as the sequence maintains the abihty to interact with RepA and display
CIS activity. Minor variations, such as substitutions oi deletions within the cis sequence are also contemplated such as modifications at 1, 5, 10 up to 20 nucleotides within the wildtype cis sequence.
The cis element is required for cis activity of the repA protein (Praszkier and Pittaid 1999 J. Bacteriol 181. 2765-2772) The cis DNA element should therefore also be located 3' in the DNA construct to the DNA encoding the lepA sequence On leachmg the cis sequence, the RNA polymerase will be paused, allowing the encoded ' protein to bind the DNA target sequence
In one embodiment of the present invention, the DNA binding protein itself comprises RepA or a fragment oi variant thereof capable of DNA binding, including at least the 20 C-termmal ammo acids of RepA capable of binding to the cis DNA element. In this embodiment, the DNA target sequence comprises an on sequence, for example the onR sequence, In alternative aspects of the present invention, the DNA binding protein is provided by an alternative protein with the relevant DNA target sequences recogmsed by such binding protein incorporated mto the sequence, In each of these embodiments, DNA-protein binding is direct in that the peptide encoded by the DNA construct will bind directly to the encoding DNA construct In alternative aspects of the invention, as described in more detail below, the DNA-protein binding may be indirect through the use of a peptide tag-DNA tag, bifunctional agent and/or suitable linkers
In one aspect, the same sequence may therefore provide both the peptide capable of binding the DNA target sequence and the C terminal ammo acids of repA. Such a sequence may be oi may comprise a complete lepA sequence, or a fragment or variant thereof of a repA sequence which letains the ability to bind to the DNA target sequence used Where the repA acts as a DNA binding protein, both cis and oil sequences (Praszkier and Pittard 1999 J Bacteriol 181: 2765-2772) are required for cis activity (cis) and DNA binding (on) In this aspect, therefore, the DNA target sequence is an on sequence and the peptide or protein capable of binding said target is a repA protein. The position of on in the DNA constructs of the invention may be vaued. As described earlier, suitable repA, cis and on sequences may be provided by one or more plasmids. For example, suitable sequences may be provided from the
IncI complex plasmids or the hicF, hrcB, hicK, IncZ and LicL/M plasmids The DNA sequence of the on from the Rl plasmid of hicll is given m SEQ ID NO: 18 Tins sequence, or a fragment thereof may be included in a DNA construct of the mvention A DNA construct of the invention may include a complete ori sequence or may include a fragment thereof which is capable of being bound by the repA protein being used
The RepA protein used in accordance with the present mvention may also comprise a fragment or variant of RepA, so long as such variant or fragment of RepA maintains the ability to bmd to the selected on sequence Such variant or fragment of RepA may include substitutions, for example, at 1, 2, 3 up to 20 ammo acids within the RepA sequence so long as such variants maintain the ability to bind to the ori sequence A suitable fragment of RepA is an on binding sequence of RepA. Ori sequences include those which are present m wild type plasmids as described above Typically, such an on sequence is 170 to 220 nucleotides in length. Fragments and variants of wild type on sequences may also be used, so long as such on sequences maintain the ability to be recognised by RepA. Further cis acting members of the RepA protein family can be used For example, the RepA family of proteins is found on plasmids with a broad host range i.e. one RepA plasmid may be found in different bacterial species Isolation of a repA family plasmid from (for example) a thermophilic, sulfopliic, halophilic or acidophilic bacterium, would provide repA-cis-on DNA that could be used under the current invention at elevated temperatures 01 extremes of salt, pH or sulphur concentrations Such members of the RepA family would be advantageous in isolating library members that can bind to target molecules undei such extreme conditions. Suitable on sequences for use in combination with selected RepA proteins can readily be determined by monitoring for the interaction of RepA with such an on sequence
The basic principle of the invention may therefore be described with reference to the repA/cis/ori system, as shown m Figure 1 This shows an example of a DNA constnict of the invention. This construct comprises, from 5' to 3', apromoter sequence, a sequence encoding a library member peptide, a sequence encoding a repA protein, a cis DNA element and an on sequence Briefly, the DNA sequence is
transcribed from the promoter by RNA polymerase to RNA The rho dependent termination function present m the cis DNA element causes the RNA polymerase to pause at this part of the sequence. This allows the repA protein and the library peptide to be translated. The repA protein is then able to bind to the on sequence, linlcing the encoded protein to the encoding DNA construct.
In one preferred embodiment, library raembei DNA sequence(s) are fused to the lepA, cis and on DNA of the hicFII plasmid Rl (Masai H et al 1983 Pioc Natl Acad Sci USA 80 6814-6818) In this embodiment, the library member DNA sequence(s) of interest may be joined by a region of DNA encoding a flexible amino acid hnlcer, to the 5'-end of the repA DNA, under the control of an appropriate promoter and translation sequences for in vitro transcription/translation. Many suitable promoters are known to those skilled in the art, such as the araB, tac promoter or the T7, T3 or SP6 promoters, amongst others The promoter should be upstream of the polypeptide sequence to be expressed.
The repA family of proteins is used herein by way of example, not limitation. Other umrelated non-covalently binding cis acting DNA binding proteins could be used in this invention
In a further embodiment, non-cis acting DNA binding proteins may be converted to having cis-activity (see Figuie 2) This may be achieved by using such proteins, capable of binding the DNA target sequence, either directly or indirectly, m combination with sequences which can confer cis-activity upon them. Cis activity may be conferred on a binding protein that does not normally act in cis by including in the DNA construct a DNA element that directs cis-activity such as the cis element of the repA system. Such an element may be included to ensure that the DNA binding by the DNA binding protein is cis, that is, an encoded DNA binding protein will bind to the DNA construct from which it has been transcribed and translated. In one embodiment, a suitable DNA construct may therefore comprise the DNA element that directs cis-activity (the cis DNA element) from the repA system Such an element may further comprise DNA encoding a portion of the C-terminal end of RepA, preferably at least 20 ammo acids, more preferably 30 amino acids, up to 40, 50, 60 or 70 amino acids from the C-tennmal portion of lepA, wherein said
fragment of repA is capable of interacting with the DNA element within the construct In a furthei example, piotems such as the cis acting transposases, Tn5 and IS 903, amongst others, could be used under the cunent invention (McFail E. J Bacteriol 1986 Aug 167.429-432; Derbyshire KM & Grindley ND. Mol Microbiol. 1996 Sep 1.1261-72.). DNA encoding sequences of the present invention may comprise wild type sequences encoding the desired fragment of RepA, degenerate sequences encoding fragments of wild type RepA or sequences encoding variants of such fragments of RepA which maintain the ability to interact with the cis element incorporated into the DNA construct. Such variants may include substitution of 1, 2, 3 or 4 amino acids within the 20 ammo acid C-termmal of RepA
The repA family of proteins is used herein by way of example, not limitation. Any DNA element capable of conferring cis-activity on a non-cis acting protein could be used .
.Any non-cis acting protein may be converted in tins way By way of example, not exclusion, the estrogen receptor DNA binding domain (DBD) can be converted into a cis acting DNA binding protein. The oestrogen receptor DNA binding domain fragment (amino acids 176-282) has been expressed in E coli and shown to bind to the specific double stranded DNA oestrogen receptoi target HRE nucleotide sequence, with a similar affinity (0.5nM) to the parent molecule (Murdoch et al. 1990, Biochemistry 29: 8377-8385, Mader et al., 1993, DNAs Research 21: 1125-1132) In one embodiment, the DNA encoding this sequence is fused, preferably at the 3'-end, to the DNA encoding at least the last 20 ammo acids of rep A, the cis DNA element, and the DNA up to the on sequence followed by the estrogen receptor tai-get recognition sequence (5'-TCAGG TCAGA GTGAC CTGAG CTAAA ATAAC AC ATT CAG-3\ SEQ ID NO- 14) which leplaces the repA on recognition sequence. The DNA sequence(s) of mteiest may then be joined by a region of DNA encoding a flexible ammo acid Imlcer, to the 5'-end of to the estrogen receptor DNA fragment, under the control of an appiopnate promoter and translation sequences for in vitro transcription/translation. Expression of this polypeptide directs the estrogen receptor DBD to its target sequence, present m place of the noimal on sequence, on the DNA encoding that polypeptide. Protem-DNA complexes can then be isolated by
capture on a target protein Unbound protem-DNA complexes can be washed away, allowing enchment for DNA encoding peptides or proteins of interest, which can then be recovered by PCR, and enriched further by performing several further Cycles of in vitro expression and protein-DNA complex capture using methods described previously
It will be clear that this approach will apply to other DNA binding proteins simply by using the cis DNA element and a sequence encoding at least the C-termmal 20 amino acids of rep A, or equivalent elements from a different cis-acting system in the DNA constructs.
In another embodiment, libraries of randomized DNA binding proteins, such as zinc finger proteins, helix-loop-hehx proteins or hehx-turn-helix proteins by way of example, may be screened for specific binding to a target sequence of interest (see Figure 3) In this embodiment, the ori recognition sequence of repA may be replaced by a target sequence of interest, and the majority of the rep A coding sequence by a library of randomised zinc finger pioteins. The DNA binding proteins therefore act as both the library member peptides and the proteins capable of binding the DNA target sequence m this aspect. The DNA encoding each zinc finger protein, may additionally be joined, at the 5'-end, to a peptide tag sequence which can be recognized by an another capture protein such as an antibody, and at the 3'-end, to the DNA encoding at least the last 20 ammo acids of repA, the cis DNA element, and the DNA up to the ori sequence followed by the target sequence of interest. Expression of this polypeptide directs the zinc finger protein to the target sequence of interest, present in place of the normal on sequence, on the DNA encoding that polypeptide Binding to the target sequence will only occur if the randomised zinc finger domain is capable of binding to the sequence of interest Protein-DNA complexes can then be isolated by capture with a binding protein which recognizes the peptide tag at the N-termmus of the fusion protein polypeptide. Unbound DNA can be washed away, allowing enhanment for DNA encoding zinc finger proteins capable of binding the target sequence, which can then be recovered by PCR, and enriched further by performing several further cycles of in vitro expression and protein-DNA complex capture.
As explained above, the binding peptide may bind diiectly to the DNA target sequence, for example m the case of a DNA binding protem-target sequence pair, or It may bind indirectly to the DNA target sequence, for example via a bifunctional agent and optionally a DNA tag (see Figure 4):
In one embodiment, DNA encoding a peptide tag which is not able to bind directly to the DNA target sequence is joined to the 5'-end of library member DNA sequence(s) of interest, optionally by a region of DNA encoding a flexible amino acid hnlcer, under the control of an appropriate promoter and translation sequences for in vitro transcription/translation This forms the DNA encoding the binding peptide, as the encoded peptide is linlced indirectly to the DNA target sequence Optionally at the 3'-end of the library member DNA sequence is the DNA encoding at least the last 20 amino acids of repA and the cis DNA element, but not the on target sequence of lepA The DNA target sequence may be or may comprise a DNA tag. Such a DNA tag may be a single modified base. for example, when preparing the library DNA construct contaimng the elements described, the DNA may be tagged at the 3'-end with, by way of example not limitation, molecules such as fluorescein or biotin.
Prior to in vitro expression, the library DNA fragments may be mixed with a bifunctional agent, one function of which is to recognize and bind to the target sequence which may be at the 5' end of the DNA, m a ratio of one DNA fragment, one bifunctional molecule The other functional element of tins bifunctional agent is a binding agent that can lecognize and bind to the peptide tag which may be encoded at the 5'-end of tire DNA fragment By way of example not exclusion, the bifunctional agent can be composed of an Fab fragment recognizing the fluorescein or biotin tag on the DNA, and anothei Fab fragment recognizing the peptide tag encoded in the DNA. It is clear to those skilled in the art that this bifunctional agent can be made by many different methods such as chemically cross-lmking the two elements, or by expressing the two elements as a fusion protein, or as a bi-specific antibody Said methods of creating a bifunctional agent are given by way of example not exclusion
The bifunctional agent may be bound to the DNA construct prior to expression of the encoded peptide or may be provided during expression.
The fusion protein is then transcribed and translated from the DNA construct while bound to the bifunctional agent. The peptide tag is translated first, and can be bound by the second element of the bifunctional agent, prior to release of messengei RNA or RNA polymerase from the DNA. This creates a functional protein-DNA complex where both expressed polypeptide and DNA encoding that peptide are linked through the bifunctional agent The peptide tag molecule is therefore linked indirectly, but specifically, to the DNA target (tag). By linking the protein to the DNA construct in tins way, it is possible to screen for a protein having paiticular properties, as described below, and then to identify the encoding DNA which is linked to that protein. By using a bifunctional agent rather than covalent binding between the protein and DNA, the DNA construct may be more easily separated form the protem without the risk of damaging the DNA
Protein-DNA complexes can then be isolated by capture of a target protein Unbound protein-DNA complexes can be washed away, allowing enrichment for DNA encoding peptides or proteins of interest, which can then be recovered by PCR, and enhched further by performing several further cycles of in vitro expression and protein-DNA complex capture using methods described previously.
Additionally, under this embodiment, the DNA can be bound directly, for example by covalent binding, to a bifunctional agent such as a polymer. Such a polymer can contain more than one binding element that could recognise the peptide tag, allowing multivalent display of a peptide expression library molecule in a unit containing the DNA encoding the displayed peptide By way of example, not limitation, said polymers can be composed of polyethylene as well as other polymeric compounds, capable of being fused to DNA The DNA construct of the invention may therefore be provided bound to such a bifunctional agent, or bound to a DNA tag as decsribed above which is capable of being bound by such a bifunctional agent
In all embodiments of the invention, the DNA constructs include appropriate promoter and translation sequences for in vitro transcription/translation Any suitable promoter can be used, such as the ara B, tac promoter, T7, T3 or SP6 promoters amongst others The promoter is placed so that it is operabiy Irnlced to the DNA sequences of the invention such that such sequences are expressed
The DNA encoding the library member peptides may be produced by any souicible means. In paiticular, such DNA may comprise DNA isolated from cDNA, obtained by DNA shuffling, and synthetic DNA.
The DNA construct may also encode ammo acid linkers within the expressed fusion protein In Particular, a flexible amino acid linker may be included to join the DNA binding peptide/RepA to the library member peptide
According to the invention, with leference to this preferred embodiment, peptide or protem expression libraries, linked to the DNA encoding them, can be generated and peptides with the desired activity selected by the following steps.
Constructing a library of fusion proteins
A DNA library of peptides or proteins may be fused to DNA encoding a peptide capable of binding to the DNA target sequence, such as a cis acting DNA binding protein DNA, by a region of DNA encoding a flexible ammo acid Imlcer, under the control of an appropriate promoter and with a translation, or ribosome binding site, start and stop codons, m a manner suitable for in vitro expression of the peptide library members and binding proteins In the example of the repA protein, the DNA (such as DNA) library members are fused to the repA DNA binding protem DNA, or a fragment thereof The cis and on sequences may be included in the construct downstream of the other elements. In the case of a DNA library, said DNA constructs are designed to be suitable for in vitro transcnption and translation.
Expression and cis binding of DNA library fusion proteins
In order to allow cis activity, a coupled bacterial transcription/translation environment such as the S30 extract system (Zubay, G. 1973 Aim. Rev. Genet. 7: 267) may be used Expression of the peptide, such as the DNA library member peptide-repA fusion protem, in this environment, will result in binding of the fusion protein to the DNA encoding that fusion protein, provided that both cis and on sequences are present. When libraries of peptide-repA fusion proteins are expressed in this manner, this process results in the production of libraries of protein-DNA complexes where the protein attached to the DNA is encoded by that fragment of
DNA from which it was expressed, thereby allowing subsequent selection of both peptides oi protein of interest, and the DNA encoding said peptides. The complexity of these libraries is enhanced by the in vitro natuie of the method, libraries of at least 1010-1014 DNA fragments, if not even larger libraries, can easily be generated.
Compounds that prevent nuclease activity, or reduce non-specific DNA-protem or protern-protein interactions may be added duimg this transcuptionytranslation reaction and cis-bmding Examples of suitable compounds include detergents and blocking protems such as bovine serum albumin (BSA)
Selection of the peptide of interest
An in vitro peptide expression library produced by a method of the present invention may be used to scieen for particular members of the library for example, the library may be screened for peptides with a Particular- activity or a particular binding affinity Protein-DNA complexes of interest may be selected from a library by, for example, affinity or activity enrihment techniques. This can be accomplished by means of a ligand specific for the protein of interest, such as an antigen if the protein of interest is an antibody. The hgand may be presented on a solid surface such as the surface of an ELISA plate well, or in solution, for example, with biotinylated ligand followed by capture onto a streptavidin coated surface or magnetic beads, after a library of protein-DNA complexes had been incubated with the ligand to allow ligand-hgand interaction Following either solid phase or in solution incubation, unbound complexes are removed by washing, and bound complexes isolated by disrupting ligand-ligand interactions by altering pH in the well, 01 by other methods laiown to those skilled m the art such as protease digestion, or by releasing the DNA directly from the complexes by heating or phenol-chloroform extraction to denature the lepA-on DNA binding DNA can also be released by one of the methods above, dnectly into PCR buffer, and amphfied. Alternatively, DNA may be PCR amplified directly without lelease from the complexes Optionally, DNA not bound by the binding for example repA protein, can be protected from degradation by non-specific DNA binding proteins such as histones, by way of example. It will be clear to one skilled in the art that many othei
non-specific DNA binding proteins could be used for this purpose Further, compounds that prevent nuclease activity, oi reduce non-specific DNA-protein or protein-protein interactions may be present during the selection process. Examples of suitable compounds include detergents, blocking proteins such as found m milk powder or bovine serum albumin (BSA), heparin or aurintricarboxylic acid.
Recovering bound complexes, reamplifying the bound DNA, and repeating the selection procedure provides an enrichment of clones encoding the desired sequences, which may then be isolated for sequencing, further cloning and/or expiession For example, the DNA encoding the peptide of interest may be isolated and amplified by, for example PCR. In one embodiment, repeated rounds of selection and DNA recovery may be facilitated by the use of sequential nesting of PCR primers DNA ends are generally damaged after multiple PCR steps. To lecover DNA from such damaged molecules required the primers to be annealed away from the ends of the DNA construct, thereby sequentially shortening the construct with every round of selection.
In one aspect, the DNA construct and/or the encoded protein may be configured to include a tag. Such a peptide or DNA tag, for example as described above, may be used in the separation and isolation of a library member of interest Such a tag may also be used to hold the library members, for example on a solid support for use in the screening methods described herein.
It can therefore be seen that the screening methods of the present invention may include the further step of selecting and isolating the relevant library member peptide, allowing the peptide exhibiting the desired properties, and also the DNA encoding that peptide, to be identified and purified
The invention therefore encompasses peptides and DNAs that have been identified by a metliod of the invention. These peptides and DNAs may be isolated and/or purified The peptides or DNAs isolated by a method of the invention may be modified, for example by deletion, addition or substitution of ammo acids or nucleotides. Suitable modified peptides or DNAs may show at least 50%, at least 75%, at least 90%, at least 95% or more amino acid or nucleotide sequence identity to the peptide or DNA isolated by the metliod of the invention. Peptides identified by
a method of the invention may be modified for delivery and/or stability purposes for example, such peptides may be pegylated (attached to polyethj'lene glycol) to piolong serum half life or to prevent protease attack. Peptides identified by a method of the invention may be modified m other display systems such as phage display or by synthesising and screening peptide variants. A collection of such modified sequences may form a new library which may be incorporated into constructs of the invention and further screened to find, for example, a variant sequence showing improved binding to a particular ligand Thus in one embodiment, a library of peptides for use in the methods of the invention may be a library of structurally related peptides
Alternatively, a library of essentially random peptide sequences may be used Numerous types of libraris of peptides fused to the cis acting DNA-binding protein can be screened under this embodiment including •
(i) Random peptide sequences encoded by synthetic DNA of variable lengtli (ii) Antibodies or antibody fragments, for example single-chain Fv antibody fragments. These consist of the antibody heavy and light chain vaiiable region domains joined by a flexible linker peptide to create a single-chain antigen binding molecule
(ill) Random cDNA fragments of naturally occuring proteins isolated from a cell population containing an activity of interest.
(iv) Random peptide sequences inserted into, or replacing a region of a known protein, whereby the known protein sequence acts as a scaffold, which constrains the random peptide sequence. Many such scaffolds have been described, by way of example, not exclusion, CTLA-4 (WO 00/60070), has been used as a scaffold for peptide libraries.
In another embodiment the invention concerns methods for screening a DNA library whose members require more than one chain for activity, as required by, for example, antibody Fab fragments for ligand binding In this embodiment heavy or light chain antibody DNA is joined to a nucleotide sequence encoding a DNA binding domain of, for example, lepA. Typically the unknown antibody DNA library sequences for either the heavy (VH and CHI) or light chain (VL and CL) genes are
inserted m the 5' region of the rep A DNA, behind an appropriate promoter and translation sequences Thus, repA fused to a DNA hbrary member-encoded protein is produced bound to the DNA encoding that protein The second known chain, encoding either light or heavy chain protein, is expressed separately either:
(a) from the same DNA fragment containing the repA and the first polypeptide fusion protein library, or
(bj from a separate fragment of DNA present in the in vitro transcription/translation reaction
The laiown chain associates with the library of unknown fusion proteins that are fused to the lepA protein and thereby bound to the DNA for the unknown chain The functional Fab library can then be selected by means of a ligand specific for the antibody
The DNA identified by a screening method of the invention, e g the DNA encoding the selected library member peptide, may be cloned into a vector. In one embodiment, the DNA identified by a method of the invention is operably linlced to a control sequence which is capable of providing for the expression of the codmg sequence by the host cell, i.e the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in then intended manner. A regulatory sequence, such as a promoter, "operably linlced" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence
Such expression vectois aie loutinely constructed m the arl of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned m the correct orientation, m order to allow for protein expression Other suitable vectors would be-apparent to persons skilled in the art. By way of further example m this regard we lefer to Sambrook et al 1989
The vectors may be for example, plasraid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said DNA
and optionally a legulator of the promotei The vectors may contain one or more selectable marker genes, for example an ampicillm resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or tiansform a host cell, for example, a mammalian host cell The vectors may also be adapted to be used in vivo, for example m a method of gene therapy.
Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed For example, yeast promoters include S cerevisiae GAL4 and ADH piomoters, S pombe nmt\ and adh promoter. Mammalian promoters include the metallothionem promoter which can be induced in response to heav}' metals such as cadmium Viral promoters such as the SV40 large T antigen promoter oi adenovirus promoters may also be used. All these piomoters are readily available in the art
Mammalian promoteis, such as ß-actin promoters, may be used Tissue-specific promoters are especially preferred Viral promoters may also be used, for example the Moloney murine leulcaema virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE piomoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR) Viral promoters are readily available m the art
The vector may further include sequences flanJcing the polynucleotide of interest giving lise to polynucleotides which comprise sequences homologous to eulcaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination In Particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell Other examples of suitable viral vectors include herpes simplex viral vectors and letrovinuses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses Gene transfer techniques using these viruses are known to those sldlled in the art.
Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome Replication-defective adenovirus vectors by contiast remain episomal and therefore allow transient expression
Such expression vectois may be used to identify ligands of interest, i.e. molecules that bind to the peptide library member by standard binding assays such as ELISA, or enzymatic assays where appropriate substrates give, for example a colour-change, light emission or fluorescence Other functional assays could be used, where available
In an alternative embodiment, a DNA identified ny a method of the invention may be cloned into a non-expression vector Such a vector may be used to further characterise the DNA, for example by sequencing
Alternatively, hgands of interest may be identified without cloning. Examples of suitable methods include the in vitro expression of individual DNA sequences recovered from a screening method of the invention, and sequencing of individual DNAs recovered from such a screening method Such individual DNA sequences may optionally be amplified
The invention also includes cells that have been modified to express a peptide identified by a method of the invention, for example by introducing an expression vectoi as described above into the cell. Such cells include transient, or preferably stable highei eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modified by insertion of vectors encoding for a peptide identified by a method of the invention include mammalian HEK293T, CHO, HeLa and COS cells Preferably the cell line selected will be one which is not only stable, but also allows for mature glycosydation and cell surface expression of the peptide Expression may be achieved in transformed oocytes. A peptide identified by a method of the invention may be expressed m cells of a transgenic non-human animal, preferably a mouse. A peptide identified by a method of the invention may also be expiessed mXenopus laevis oocytes or melanophores.
In order that the invention is more fully understood, embodiments will now be described in more detail by way of example only and not by way of limitation with reference to the figures below.
Examples of some of the embodiments of the invention are given below
Materials and Methods
The following proceduies used by the present applicant aie described m Sambrook, J., et al., 19S9 supra., analysis of restriction enzyme digestion products on agarose gels, DNA purification using phenol/chloroform stock solutions, preparation of phosphate buffered saline
General purpose leagents were purchased from SIGMA-Aldnch Ltd (Poole, Dorset, U.K ) Oligonucleotides weie obtained from SIGMA-Genosys Ltd (Cambridgeshire, U K.) Ammo acids, and S30 extracts were obtained from Promega Ltd (Southampton, Hampsliiie, U.K.). Deep Vent and Taq DNA polymerases were obtained from New England Biolabs (Cambridgeshire, U.K.). Taqplus DNA polymerase was obtamed from Stratagene Inc. (Amsterdam, Netherlands). GeneClean DNA gel purification kits were obtained from BIOl 01 (La Jolla, California, U.S.A.), anti-human IgK antibodies from Imnunologicals Direct Ltd (Oxfordshire, U K.), anti-c-myc polyclonal from Vector Labs Inc (Cambridgeshire U.K.), and anti-V5 antibody from Abeam Ltd (Cambiidgeshire U K.). Superblock bloclang agent was obtamed from Perbro Science (Cheshire, U.K ).
Example 1. Isolation of specific cis acting nrotein-DNA complexes
The in vitro expression constructs were prepaied by sequentially adding the TAC promoter, the c-myc epitope, either the human kappa constant legion or the V5 epitope to the RepA-CIS-ORJ region, by PCR amplification Such constructs can be prepared by many methods laiown to one skilled in the ait, for example, by amplifiymg different fragments of DNA followed by assembly PCR In this example, the initial amplification template was the Rl plasmid which contains the RepA-CIS-ORI region (Masai, H. and Ai-ai, K.(198S) DNAs Res 16, 6493-6514).
(a) Primar-3' amplification The RepA-CIS-ORI region was PCR amplified
from a single colony of the strain ECO K12 harbourmg plasmid Rl using 12.5pmoI
of each of the primers REPAFOR (SEQ ID 01) and ORIREV (SEQ ID 02) in a 50)xl
reaction containing 0.25mM dNTPs, 2.5 units Taqplus Precision DNA polymerase,
Ix PCR reaction buffer (Stratagene Inc, Amsteidam, Netherlands). The REPAFOR
pnmer anneals to the 5'-end of the RepA coding region The ORIREV primer
amieals to the 3'-end of the non-codmg ORI region
PCR reactions were cained out on a Eppendorf Master Cyclei for 1 cycle of 4 minutes and 15 seconds at 94°C followed by 30 cycles of 94°C, 45 seconds; 60°C, 45 seconds, 72°C, 45 seconds, followed by a single cycle 10 minutes at 72'C. Reaction products were electiophoresed on an agarose gel, excised and products purified from the gel into 40)al steule water using a Geneclean II kit according to the manufacturers instructions (BiolOl, La Jolla, California, U S.A )
(b) Secondary amplification. One µl (500 pg) of 100 times diluted gel-purfied
primary reaction product was re-amplified using 12.5pmol of each of the primers
CKREPFOR (SEQ ID 03) and ORIREV (SEQ ID 02) m a 50µl reaction containing
0.25mM dNTPs, 2.5 umts Taqplus Precision DNA polymerase, and Ix PCR reaction
buffei (Stratagene Inc, Ansterdam, Netherlands). The CKREPFOR primer amieals to
the 5'-end of the primary reaction product and appends the 3' part of the kappa
constant region DNA. The ORIREV primer anneals to the 3'-end of the primary
leaction product
PCR reactions were carried out on a Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94'C, 45 seconds, 60°C, 45 seconds, 72°C, 2 minutes, followed by a single cycle 10 minutes at 72'C Reaction products were electiophoresed on an agarose gel, excised and products purified fioni the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers instructions (Biol0l, La .lolla, California, USA)
(c) Third amplification One JJ.1 (500 pg) of 100 times diluted gel-purified
primary reaction product was re-amphfied using 12.5pmol of each of the primers
V5REPF0R (SEQ ID 04) and ORIREV (SEQ ID 02) m a 50µ1 reaction contaimng
0.25mM dNTPs, 2.5 units Taqplus Precision DNA polymerase, and Ix PCR reaction
buffer (Stratagene Inc, Amsterdam, Netherlands). The V5REPF0R primer anneals to the 5'-end of the primary leaction product and appends the 3' part of the V5 epitope DNA. The ORIREV primer anneals to the 3'-end of the primary leaction product.
PCR reactions were carried out on a Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94°C, 45 seconds, 60°C, 45 seconds, 12°C, 2 minutes, followed by a single cycle 10 minutes at 72°C Reaction products were electrophoresed on an agarose gel, excised and products purified fiom the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers instructions (Biol01, La .ToUa, California, U S.A )
(d) Fourth amplification One µl (500 pg) of 100 tunes diluted pCKV5
plasmid using 12.5pmol of each of the primers MYCCKFOR (SEQ ED 05) and
CKREV (SEQ ID 06) in a 50µx1 reaction containing 0 25mM dNTPs, 2.5 units
Taqplus Precision DNA polymerase, and Ix PCR leaction buffer (Stratagene Inc,
Amsterdam, Netherlands) The pCKV5 plasmid contains the human kappa constant
legion cDNA (McGregor DP, Molloy PE, Cunmnghara C, & Harris WJ. 1994 Mol
hiimunol. 31: 219-26) and the V5 epitope DNA (Southern JA, Young DF, Heaney F,
Baumgartner WK, Randall RE. 1991 J. Gen. Virol. 72: 1551-7). The MYCCICFOR
primer anneals to the 5'-end of the kappa constant legion DNA and appends the 3'
part of the MYC epitope DNA. The CKREV primei anneals to the 3'-end of the
kappa constant region DNA
PCR reactions were earned out on a Eppendoif Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94'C, 45 seconds; 60'C, 45 seconds, 72°C, 2 minutes, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile water using a Geneclean II kit according to the manufactuiers mstiuctions (BiolOl, La Jolla, California, USA)
(e) Fifth amplification One µl (500 pg) of 100 times diluted pCKV5 plasmid
using 12 5pmol of each of the primers MYCV5F0R (SEQ ID 07) and V5REV (SEQ
ID 08) in a 50µ1 reaction containing 0.25mM dNTPs, 2 5 units Taqplus Precision
DNA polymerase, and Ix PCR reaction buffer (Stratagene Inc, Amsterdam,
Netherlands). The MYCV5F0R primer anneals to the 5'-end of the V5 epitope DNA
and appends the 3' part of the MYC epitope DNA. The V5REV primer anneals to the 3'-end of the V5 epitope DNA.
PCR reactions were carried out on a Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 Cycles of 94°C, 45 seconds; 60°C, 45 - seconds; 72°C, 30 seconds, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoresed on an agaiose gel, excised and products purified fiom the gel into 40µ1 sterile water using a Geneclean II kit according to the manufacturers ' instructions (BiolOl, La Jolla, California, U.S A ).
(f) Sixth amplification. One µl (500 pg) of 100 times diluted pTACPZA
plasmid (ref) using 12.5pmol of each of the primers TAC3 (SEQ ID 09) and
MYCTACREV (SEQ ID 10) m a 50µl reaction containing 0.25mM dNTPs, 2.5 units
Taqplus Precision DNA polymerase, and Ix PCR leaction buffer (Stratagene Inc,
Amsterdam, Netherlands) The TAC3 primer anneals to the 5'-end of the TAG
promoter DNA. The MYCTACREV primer anneals to the 3'-end of the TAG
promoter DNA and appends the 5' part of the MYC epitope DNA.
PCR reactions were carried out on a Eppendorf Mastei Cycler for I cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94°C, 45 seconds; 60°C, 45 seconds; 72°C, 30 seconds, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers instructions (Biol0l, La Jolla, California, U.S A.).
(g) First assembly PCR. One p.! (50 ng) of each of the reaction products in (f)
and (d) using 50 pmol of each of the primers TAC5 (SEQ ID 11) and CKREV (SEQ
ID 06) in a 50µl reaction containing 0 25mM dNTPs, 2.5 units TaqDeepVent DNA
polymerase mixture (20. 1), and Ix PCR reaction buffer (New England Biolabs,
Beverly, MA, USA) The TAC5 pnmer anneals to the 5 "-end of the reaction
pioduct (f) and adds 20 nucleotides. The CKREV primer anneals to the 3'-end of the
reaction product (d)
PCR reactions were carried out on a Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94°C, 45 seconds, 60'C, 45 seconds, 72°C, 45 seconds, followed by a single cycle 10 minutes at 72°C. Reaction
products were electiophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile watei using a Geneclean II kit according to the manufacturers insti-uctions (Bio 101, La Jolla, Califorma, U.S.A).
(h) Second assembly PCR. One µl (50 ng) of each of the reaction products m (f) and (e) using 50 pmol of each of the primers TAC5 (SEQ ID 11) and V5REV (SBQ ID 08) in a 50µl reaction containing 0.25mM dNTPs, 2 5 units TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR leaction buffer (New England Biolabs, Beverly, MA, U.S.A ). The TAC5 primer anneals to the 5'-end of the leaction product (f) and adds 20 nucleotides The V5REV primei armeals to the 3'-end of the reaction product (e)
PCR leactions weie earned out on a Eppendorf Master Cycler for 1 C3'cle of 2 minutes and 15 seconds at 94'C fohowed by 30 cycles of 94^, 45 seconds; 60°C, 45 seconds, 72°C, 45 seconds, followed by a single cycle 10 minutes at 72'C. Reaction products were electrophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers mstructions (Biol0l, La Jolla, California, U S.A.).
(i) Third assembly PCR. One µl (50 ng) of each of the reaction products in (b) and (g) or using 50 pmol of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) m a 50µ1 reaction containing 0.25mM dNTPs, 2.5 umts TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR reaction buffer (New England Biolabs, Beverly, MA, U.S.A ). The TAC3 primer aimeals 20 nucleotides downstream to the 5'-end of the reaction product (g). The ORIREV primei amieals to the 3'-end of the reaction product (h). The reaction product in (i) is called TAC-MYC-CK-REPA-CIS-0RI(SEQID12)
PCR leactions were carried out on a Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94°C followed by 30 cycles of 94'C, 45 seconds; 60°C, 45 seconds, 72'C, 1 minute, followed by a single cycle 10 minutes at 72°C Reaction products weie electrophoiesed on an agarose gel, excised and products purified from the gel into 40)j.l sterile water using a Geneclean 11 kit according to the manufacturers mstructions (Bio 101, La Jolla, Califorma, U.S A )
(j) Fourth assembly PCR One µl (50 ng) of each of the reaction products m (b) and (h) or using 50 pmol of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) in a 50µl reaction containing 0.25mM dNTPs, 2.5 units TaqDeepVent DNA polymerase mixture (20:1), and Ix PCR reaction buffer (New England Biolabs, Beverly, MA. U.S.A.). The TAC3 primer anneals 20 nucleotides downstream to the 5'-end of tihe reaction pioduct (g) The ORIREV primer amieals to the 3'-end of the reaction product (b) The leaction product m (i) is called TAC-MYC-V5-REPA-CIS-0RI (SEQ ID 13)
PCR reactions were earned out on a Eppendorf Mastei Cycler for 1 cycle of 2 minutes and 15 seconds at 94'C followed by 30 cycles of 94'"C, 45 seconds; 60"'C, 45 seconds; 72°C, 1 minute, followed by a single cycle 10 minutes at 72°C Reaction products were electiophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers instructions (Biol01, La Jolla, California, USA)
Preparation of in vilro transcription/translation reaction. The reaction was set up on ice, using a Promega bacterial linear template S30 coupled in vitro trarscription/translation reaction kit as follows.
20µl TAC-MYC-CK-REPA-CIS-ORI template (0.5µg of final construct DNA SEQ ID 012 above); 20µlTAC-MYC-V5-REPA-CIS-0RI template (0 5µg of final constiuct DNA SEQ ID 013 above): 20µl complete amino acid mix (Promega), 80µl 830 Premix; 60µl S30 mix,
and the reaction was allowed to proceed at 25°C for 30 minutes and placed on ice, then diluted 10 fold with blocking buffer (Superblock (Perbio Ltd), 0.1 % Tween 20, 200µg/ml herring sperm DNA)
DNA-protem complex capture NUNC star immunotubes were coated with 10µg/ml of either anti-c-myc antibody, anti-V5 antibody, or anti-human kappa chain antibody, m 500µl PBS pei tube overnight at 4°C. An additional tube was left blank as a negative control. Tubes were washed 2x PBS and blocked for 1 hour at room temperature with Superbloclc/PBS/0.lmg/ml hening sperm DNA/ 0.1% Tween 20 and then washed 2x PBS. 500µl of diluted transcription/translation reaction was
added to each tube and incubated at room temperature for 1 hour Tubes were washed 5x PBS/0 1% Tween 20, then 1x30 mmutes with 2ml Superblock/PBS/0.lmg/ml herring sperm DNA/ 0.1% Tween 20. then 5x PBS. DNA was recovered with 300µl T.E buffer plus 300µl phenol/chloroform for 5 minutes with shaking. This was centrifuged at 13,200g for 5 minutes and DNA precipitated with 0.5 volume of 7.5M ammonium acetate, 20µg glycogen and tluee volumes of absolute etlianol Following centrifugation, pellets were washed with 70%o ethanol, vacuum dued and resuspended in 20µl water. l0µl of lecovered DNA was leamplified m 50µl reactions with TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) primers Reaction products were electiophoresed on a l%o agarose/TAE gel (Figure 5).
Example 2. Separating the RepA-DNA complex
The two in vitro expression constructs (SEQID12 and SEQID13) already described in example 1 were used in a selection experiment against anti-human C-kappa antibody as described in Example 1, except that DNA was recovered and released from RepA by using either of following methods; Glycine, Tnethylamme, Phenol/Chloroform, Proteinase K, and EDTA These methods are described below.
Glycine: tube was incubated with 500µl of 200mM Glycine, 150mMNaCl (pH2.0) for 10 minutes. The glycine eluate was then transferred to a fresh eppendorf tube and 50µl of 2M Tris (pH 8.5) added
Tnethylamine- the tube was incubated 500µl of 0.1 M Triethylamine for 10 minutes and the triethylamine eluate was then transferred to a fresh eppendorf tube and 250^1 of IM Tns (pH 7 4) added
Phenol/Chloroform, as example 1 above.
Proteinase K the tube was incubated with 500µl of l00mM Tns (pH 8 0), 10 mM EDTA (pH 8 0), 0 5% SDS for 30 minutes at 37°C The Proteinase K eluate was then transfened to a fresh eppendorf tube
EDTA the tube was incubated with 250µl of l0mM Tns (pH 8 0), 1 mM EDTA 500mM NaCl and 250µl of Phenol/Cliloroform for 5 mmutes The EDTA eluate was then transferred to a fresh eppendorf tube
After recovery of DNA the DNA was Phenol/Clilorofonn extracted, where appropriate, followed by Ethanol precipitation as described in Example 1 l0µl of resuspended DNA was reamplified in 50ul leactions with TAC3 (SEQID09) and CISREV (SEQID019) primers. The CISREV primer anneals 196 bases upstream of the binding site of ORIREV (SEQID02). Reaction pioducts were electrophoresed on a 1 % agairse/TAE gel (data not shown) Only the CK-DNA containing construct (SEQID 12) was amplified, in appioximately equivalent amounts
This not only tells us that any of the methods described above for recovering and releasing DNA from RepA can be used, but this result also suggests that RepA interacts m a non-covaleiit manner with its cognate DNA
Example 3. Detection of specific anti-V5 binders in a V5-spiking expeiment using CIS display technology
The in vitro expression constmcts were prepared by adding the TAG promoter and either the V5 epitope oi a 12-mer NNB library to the RepA-CIS-ORI region, by PCR amplification. Such constructs can be prepared by many methods known to one skilled in the art, for example, by amplifiymg different fragments of DNA followed by assembly PCR In this example, the initial amplification template was the Rl plasmid which contains the RepA-CIS-ORJ region (Masai. H. and Arai, K (1988). Nucleic Acids Res 16, 6493-6514)
(a) Primary amplification. The RepA-CIS-ORI region was PCR amplified from a single colony of the strain ECO K12 harbouring plasmid Rl using 12.5pmol of each of the primeis REPAFOR (SEQ ID 01) and ORIREV408 (SEQ ID 20) in a 50)j.l reaction containing 0.25mM dNTPs, 2.5 umts TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR reaction buffer (New England Biolabs, Beveily, MA, U S A.) The REPAFOR primer anneals to the 5'-end of the RepA coding region The ORIREV408 primer amieals to the downstream of the 3'-end of the non-coding ORI legion
PCR reactions were carried out on a Eppendorf Master Cyclei for 1 cycle of 4 minutes and 30 seconds of 94°C followed by 25 cycles of 94°C, 30 seconds, 60'C, 45 seconds; 72°C, I minute, followed by a single cycle 10 minutes at 72°C. Reaction
products were electrophoresed on an agarose gel, excised and products purified from the gel into 40\il sterile water using a Geneclean II kit accordmg to the manufacturers instructions (Bio 101, La JoUa, California, U.S.A).
(b). Secondai-y amplification. One µl (500 pg) of 100 times diluted gel-purified primaiy reaction product was re-amplified using 12.5pmol of each of the primers V5(NNB)REPF0R (SEQ ID 21) and ORIREV408 (SEQ ID 20) in a 50µl leaction containing 0.25mM dNTPs, 2 5 units TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR reaction buffer (New England Biolabs, Beverly, MA, U.S.A ) The V5(NNB)REPF0R primer anneals to the 5'-end of the pnmary reaction product and appends the V5 epitope DNA. The ORJREV408 primer anneals to the 3 "-end of the primary reaction product
PCR reactions were carried out on a Eppendorf Master Cycler for 1 cycle of 4 minutes and 30 seconds of 94°C followed by 25 cycles of 94'C, 30 seconds; 60°C, 45 seconds, 72°C, 1 minute, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoiesed on an agaiose gel, excised and pioducts purified from the gel into 40µ1 sterile water using a Geneclean II kit accordmg to the manufacturers instructions (BiolOl, La Jolla, California, Ll.S.A).
(c). Tliird amplification. One pi (500 pg) of 100 times diluted gel-purified primaiy reaction product was re-amplified using 12 5pmol of each of the primers NNBREPFOR (SEQ ID 22) and ORIREV408 (SEQ ID 20) in a 50pl reaction containing 0.25mM dNTPs, 2 5 units TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR reaction buffer (New England Biolabs, Beverly, MA, U.S.A.), The NNBREPFOR primer amieals to the 5'-end of the primary reaction product and appends a random amino acid 12-mer NNB libraiy DNA. The ORIREV408 primei amieals to the 3'-end of the primary reaction product
PCR leactions were earned out on a Eppendorf Mastei Cycler for 1 cycle of 4 minutes and 30 seconds of 94°C followed by 25 cycles of 94°C, 30 seconds, 60°C, 45 seconds; 72'C, 1 minute, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoiesed on an agarose gel, excised and products purfied from the gel into 40µl sterile watei using a Geneclean II kit according to the manufacturers instructions (BiolOl, La Jolla, California, U.S A).
(d). Fourth ainphfication. One µl (500 pg) of 100 times diluted pTACP2A plasmid (ref) using 12 5pmol of each of the primers TACFARUP (SEQ ID 23) and TACREV (SEQ ID 27) in a 50^1 reaction containing 0.25mM dNTPs, 2.5 umts TaqDeepVent DNA polymerase mixture (20:1), and 1 x PCR reaction buffer (New England Biolabs, Beverly, MA, U.S A.). The TACFARUP primer anneals to the 5'-end of the TAC piomoter DNA The TACREV primer anneals to the 3'-end of the TAC promotei DNA
PCR reactions were earned out on a Eppendorf Master Cyclei for 1 cycle, of 1 munutes and 45 seconds of 94°C followed by 25 cycles of 94°C, 15 seconds; 60°C, 30 seconds, 72°C, 30 seconds, followed by a single cycle 10 minutes at 72°C. Reaction products were electrophoresed on an agaiose gel, excised and products purified from the gel into 40)J.l sterile water using a Geneclean 11 kit according to the mairufacturers instructions (BiolOl, La Jolla, California, U S.A )
(e) First assembly PCR One jj.1 (50 ng) of each of the reaction products in
(b) and (d) using 50 pmol of each of the primers TACFARUP (SEQ ID 23) and
ORIREV408 (SEQ ID 20) in a 50µ1 reaction containing 0 25mM dNTPs, 2.5 units
TaqDeepVent DNA polymerase mixture (20-1), and Ix PCR reaction buffer (New
England Biolabs, Beverly, MA, USA) The TACFARUP primer anneals to the 5'-
end of the reaction product (d) The ORIREV480 primer anneals to the 3'-end of the
reaction product (b). The reaction product m (e) is called TAC-V5-REPA-CIS-0RI-
408 (SEQ ID 24)
PCR reactions were carried out on a Eppendorf Master Cyclei for 1 cycle of 1 minutes and 45 seconds of 94°C followed by 25 cycles of 94°C, 15 seconds; 60°C, 30 seconds; 72°C, 1 mmute and 30 seconds, followed by a smgle cycle 10 minutes at 72°C. Reaction products were electrophoresed on an agarose gel, excised and pioducts purified from the gel into 40µ1 sterile water using a Geneclean II kit accoiding to the manufacturers instructions (BiolOl, La Jolla, California, U S.A ).
(f) Second assembly PCR. One j.d (50 ng) of each of the reaction
products in (c) and (d) using 50 pmo] of each of the primers TACFARUP (SEQ ID
23) and ORIREV408 (SEQ ID 20) m a 50µl reaction containing 0.25mM dNTPs, 2.5
units TaqDeepVent DNA polymerase mixture (20 1), and Ix PCR reaction buffer
(New England Biolabs, Beverly, MA, U S.A ) The TACFARUP primer anneals to the 5'-end of the reaction product (d). The ORIREV480 primer anneals to the 3'-end of the reaction product (c)
PCR reactions were canned out on a Eppendorf Master Cycler for 1 cycle of 1 minutes and 45 seconds of 94°C followed by 25 cycles of 94°C, 15 seconds; 60'C, 30 seconds, 72°C 1 minute and 30 seconds, followed by a single cycle 10 minutes at 72 "C Reaction products were electrophoresed on an agarose gel, excised and products purified from the gel into 40µl sterile water using a Geneclean II kit according to the manufacturers instructions (Bio 101, La Jolla, California, U.S A.) The reaction product in (f) is called TAC-NNB-REPA-CIS-ORI-408 (SEQ ID 25)
Preparation of in vitro transcription/translation reaction The reaction set was set up on ice, using a Promega bacterial lineai template S30 coupled in vitro transcription/translation reaction kit as follows
20µl of 5000 times diluted TAC-V5-REPA-CIS-ORI-408 template (O.lng of final construct DNASEQ ID 24 above)
20µ1 of 5 TAC-NNB-REPA-CIS-ORI-408 template (0 5µg of final construct DNASEQ ID 25 above) 20µl complete amino acid mix (Promega) 80µl S30 Premix 60µl 830 mix
and the reaction was allowed to proceed at 25°C for 30 minutes and placed on ice, then diluted 10 fold with 2% Marvel/PBS.
DNA-protem complex capture. NUNC star imraunotubes were coated with 10µlg/ml of anti-V5 antibody m 500µl PBS overnight at 4°C. An additional tube was left blanlc as a negative control Tubes were washed 2x PBS and blocked for 1 hour at room temperatue with blocking buffei (2% Marvel, 0 1% Tween 20, 0 Img/ml hemng sperm DNA) and then washed 2x PBS 1 ml of diluted transcription/translaiton reaction was added to each tube and incubated at room temperature for 1 hour. Tubes were washed 5x PBS/0 1% Tween 20 and then 5x PBS DNA was recovered with 500µl TE buffer plus 500µl phenol/chloroform. This was centrifuged at 13,200g for 5 minutes and DNA precipitated with 1/10 volume of
3M sodium acetate, 50µg/ml glycogen and two voulmes of absolute ethanol. Following centnfugation, pellets were washed with 70% ethanol, vacuum dried and resuspended m 40µl water 20 µl of recovered DNA was reamplified in 50 µl reactions with the biotinylated primers bTAC6 (SEQ ID 26) and bCISREV (SEQ ID 19). Reaction products were electrophoresed on a 1% agarose/TAE gel.
Cloning of recovered DNA into the expression vector pDMG-K (SEQ ID 27) Reaction product were gelpurified and eiuted with 50µl sterile water using a QIAquick Gelextracation kit according to the manufacturers instructions (QIAGEN LtdWest Sussex. U.K.) Both the purified reaction product and the piasmid pDMG-K were digested with 20 units of Ncol and NotI (New England Biolabs, Beverly, MA, U.S.A). The cut piasmid was gelpurified using a QIAquick Gelextracation kit accoidmg to the manufacturers instructions (QIAGEN LtdWest Sussex, U.K ). then treated with 0 01 units of Calf Intestinal Alkaline Phosphatase (Promega, Southampton, U.K.) followed by phenol/chloioform extraction and ethanol precipitation as described above. Precipitated DNA was dissolved in 20µL of water. The cut PCR product was transferred to Streptavidm coated strips (Roche Diagnostics Ltd, East Sussex, U K.) in Ix TBS, 0.3 mg/ml BSA, 0.1% Tween 20 and incubated for 30 minutes at room temperature, shaking. This approach removes the flanlcing biotinylated DNA upstream and downstream of the Ncol and NotI site of the PCR product and enables recovery of the small DNA fragment containing the selected peptide sequence Supernatant was phenol/chloroform extracted and ethanol precipitated as described above Precipitated DNA was dissolved in 10µl of water. Cut piasmid and the isloated small DNA fragment containing the selected peptide sequence, both having Ncol and NotI overhangs, were ligated using a Quick ligation kit according to the manufacturers instructions (New England Biolabs, Beverly, MA, U.S.A) followed by phenol/chloroform extraction and ethanol precipitation as described above Precipitated DNA was dissolved in l0µl of water and electroporated into electrocompetent TGI cells according to the manufacturers instructions (Stratagene, U S.A.) and selected on plates with 2xTY, l00µg/ml ampicillin, and 2% glucose
Anti-V5 antibody ELISA screening of selected clones. 88 colomes were picked into 400µl of 2x TY, 2% glucse, and l00µG/ml ampicilin and grown overnight at 37°C, shaking 300 rpm. 50µl of the overnight cultures were transferred into 1ml of 2x TY, 2% glucose, and l00µg/ml anipicillin and grown at 37°C, shaking 300 rpm until OD 0.5 Then the cells were centrifuged at l000x g for 10 minutes. The supernatants were discarded and pellets were resuspended in 600µl of 2x TY, 0 4M sucrose, 100µg/ml ampicillin, and 1mM IPTG and grown for 4 hours at 37°C, 300 rpm. After induction the cells were centrifuged at lOOOx g for 10 minutes. 150µl of the supernatants were used in the ELISA test. NUNC Maxisoip plates were coated with l00µl of lµg/ml in Ix PBS of either anti-human kappa legion antibody or anti-V5 antibody or 50µg/ml of BSA for 7 hours at room temperatuie. An additional plate was left blanlc, only coated with PBS. Wells were rinsed 2x PBS followed by bloclang for 1 hour at room temperature with 300µl of 4% Marvel, 0.1% Tween in Ix PBS. Wells were uinsed 2x PBS, then 150µl of supernatant and 150µl of 4% Marvel, 0.1% Tween 20 in Ix PBS were added to wells and incubated for 1 hour at room temperature. Wells were then washed 2xPBS, 0.1% Tween 20 and 2x PBS Secondary antibody anti-human kappa region antibody conjugated to horse radish peroxidase (HRP)(final concentration 1 6µg/ml) was diluted 500 times in 4% Marvel, 0 1% Tween 20, Ix PBS and added to wells and incubated for 1 hour at room temperature Wells were then washed 4x PBS, 0.1% Tween 20 and 2x PBS. The HRP signal was detected by adding 200µl of TMB substrate. Reaction was stopped with l00µl of 0.5M sulphuric acid. Absorbance was lead at 450nni 35 out of 88 clones expressed well judged by HRP signal from clones screened against anti-human kappa region antibody 7 out of these 3 5 clones showed specific binding to anti-V5 antibody, thereby enriching V5-peptides from 1 in 5000 to 1 in 5, i.e. an ennchment factor of 1000 (Figure 6)
Example 4: CIS display Libran' construction, selection & screening against Bacillus slobisii
Library Construction
To generate library DNA, a promoter library DNA fragment and the RepA-CIS-ori fragment must be generated, then linked together by digestion-ligation. The tac promoter from a P2A-HA vector was used in this example, but many available promoters could be used, and ai-e well known to those skilled m the art. The initial PCR of Rep-CIS-on and TAC fragments appends Bspl20I site and the random library/Notl site respectively Two master mixes were prepared'
l0µl of 1 50 diluted P2A-HA plasmid DNA (25ng/reaction) was PCR amplified in 20x 50µ1 reaction volume containing 200µM dNTPs, IxNEB polymerase amplification buffer (l0mM KCl, l0mM (NH4)2S04, 20mM Tris-HCl pH 8.8, 2mM MgS04, 0.1% TritonX-100) with l0pmol of each of the primers TACFARUP (SEQ ID 23) and NTERMl SMER (SEQ ID 28) primers and 2 units of 20 1 Taq DNA polymerase. Deep Vent DNA polymerase mixture (NEB) for 25 cycles of 94°C, 40 seconds; 60°C, 40 seconds; 72°C, 60 seconds, followed by a 5 minutes extension at 72°C. 20µl of reaction product were electrophoresed on a 1% agarose/TAE gel and photographed, while the remainder was Qiagen column purified into 200µl water.
l0µl of Bspl20I corrected Rep-CIS-on DNA (50ng/reaction) was PCR amplified in l0x 50µl reaction volume containing 200µM dNTPs, IxNEB polymerase amplification buffer (10ml\4 KCl, lOmM (NH4)2S04, 20mM Tris-HCl pH 8 8, 2mM MgS04, 0 1% TntonX-100) with lOpmoI of each of the primers BSPREPAFOR (SEQ ID 29) and ORIREV (SEQ ID 02) primers and 2 units of 20-1 Taq DNA polymerase Deep Vent DNA polymerase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds, 72°C, 90 seconds; followed by a 5 minutes extension at 72°C 20µl of reaction product were electrophoresed on a 1% agarose/TAE gel and photographed, while tlie remaindei was Qiagen column purified into 120µl water
Library-TAC product was then digested with lOµl NotI (NEB) (l00u) for 1 hour at 37°C in a 300µl leaction volume, then Qiagen column purified into a I20)al volume of water The two products were then joined by lestriction-ligation as follows
l0xNEB buffer 4 17µ1
100mM ATP (SIGMA) 15µ1
10mg/ml acetylated BSA (NEB) 1 µl
RepA DNA 40µ1
TAC-library DNA 40µ1
Bsp 1201 (10u/µl Fermentas) 5µ1
NotI(10u/µl NEB) 5µ1
T4 DNA ligase (400u/µl NEB) 5µ1
Water 39µ1
Reaction was carried out at 37°C for two hours. 20 µ1 was assessed by gel electrophoresis, 30µl was PCR amplified directly m l0x 50µl reactions, and the remainder was gel purified and the library band excised, Qiagen column purified and PCR amplified in 20x 50µ1 reactions with primers TACFAR4 (SEQ ID 30) and ORIREV (SEQ ID 02) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds; 72°C, 90 seconds; followed by a 5 minutes extension at 72°C. DNA was gel purified in 4 Qiagen columns and the 200µl eluate pooled for ITT reactions/selection
Round 1 Selection
2 X 200µl ITT reaction was set up and incubated at loom temperature for 1 hour as follows-
REACTION 1
Library DNA 56µ1 (7µg)
2 5x buffer 80µ1
100mM methionine 2µl
S30 extract 60µ1
1ml of blocking buffer was added to each reaction (Block buffer is 4% Marvel, l00µg/ml sheared salmon sperm DNA, 0 1% Tween 20, 2 5mg/ml heparin,
in TBS), spun at 10,000g for 2 minutes, transferred to a fresh tube, then placed on ice.
100 .µl Baalliis globigii {Bg) spore suspension was washed twice with 1ml TBS/0.1% Tween 20 and was resuspended in l00µl of Block buffer This was then added to the Block buffer and allowed to bind at room temperature for 1 hour whilst mixing
The mix was then centrifuged at I6,100g for 1 minute and the spore pellet was washed six times with 1 ml of TBS/O 1% Tween 20 by mixing with a pipette and vortexing prior to centrifugation The pellet was finally washed in 1ml TBS and the supernatant was discarded
DNA was eluted by incubation of the spores m 120µl 0 5M sodium acetate pH5 5 for 10 minutes on a mixer. The spores were centrifuged at 16 JOOg for 1 minute and the supernatant was neutralised by the addition of 120µl Tris pH8 0 and then phenol/CHCl3 extracted for 5minutes at 16,100g. DNA was precipitated with 20µg carrier glycogen and two and a half volumes of ethanol. DNA was pelleted at 16,100g for 20 minutes and the pellet washed three times with 0.75ml 70% ethanol, centnfuging for 3 minutes at 16,100g in between each wash, then air dried and re-suspended in 20µl water.
10µ1 recovered DNA was PCR amplified in 10 50µl reaction with primers CISREV (SEQ ID 19) and TACFAR5 (SEQ ID 31) and 2 units of 20:1 Taq DNA polymerase: Deep Vent DNA polymerase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds; 72°C, 90 seconds, followed by a 5 minutes extension at 72°C The DNA was purified, ethanol piecipitated and re-suspended in l0µl water. 5 µ1 were further amplified by PCR using the conditions above but using the primers N0TRECREV2 (SEQ ID 32) and TACFAR5 (SEQ ID 31) for 10 cycles The product was punfied using a Qiagen PCR purification kit and eluted into 50µl 5mM Tris pH 8 0
Restriction-Ligation
This was carried out m a 30µl reaction for 1 hour at 37°C to reattach RepA-ClS-ori DNA to recovered peptides for a further round of selection
l0xNEB buffer 4 3µ1
l00mM ATP (SIGMA) 1 5µ1
10mg/ml acetylated BSA (NEB) 0 3µil
RepA DNA 2µl
TAC-library DNA 10µl
Bsp 1201 (10µl Fermentas) 1 5µl
NotI(10/µl NEB) 1 5µ1
T4 DNA ligase (400u/µl NEB) 1.5µl
Water 8 7µ1
20µ1 was PCR amplified directly in l0x 50µl reactions with primers TACFAR5 1 (SEQ ID 33) and ORIREV (SEQ ID 02) for 20 cycles of 94°C, 40 seconds, 60°C, 40 seconds, 72°C, 90 seconds, followed by a 5 minutes extension at 72°C DNA was gel purified in 1 Qiagen column and the eluate used for Round 2 of ITT reactions/selection (58µl used in R2).
Round 2
Second round selection was carried out as for round 1, with the following changes Approximately 3|a.g of input DNA were used. Block buffer was 2% bovme serum albumin, 1% gelatin, l00µg/ml sheared salmon sperm DNA, 2 5mg/ml heparin, m TBS. 10µl washed spores used in each selection Recovery PCRs used TACFAR5 2 (SEQ ID 34) and N0TRECREV2 (SEQ ID 32) primers. Finally, pull through PCR used TACFAR5 2 (SEQ ID 34) and ORIREV (SEQ ID 02) primers for 10 cycles
Round 3
Third round selection was carried out as for round 2, with the following changes' Approximately 2.5µg of input DNA was used Recovery PCRs used TACFAR6 (SEQ ID 35) and N0TRECREV2 (SEQ ID 32) primers. Finally, pull
through PCR used TACFAR6 (SEQ ID 35) and ORIREV (SEQ ID 02) primers for 10 cycles.
Round 4
Round 4 was carried out as for round 3, except that approximately 2µg of input DNA was used for the selection Recovery PCRs used TAC3 (SEQ ID 09) and N0TRECREV2 (SEQ ID 32) primers Finally, pull through PCR used TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) primers for 10 cycles
Round 5
Round 5 was carried out as for round 4
For cloning out as NcoI-NotI fragments, the stored the recovered DNA from round 5 was PCR amplified with brotmylated TAC6 (SEQ ID 26) primer and N0TRECREV2 (SEQ ID 32) Digestion with NotI was followed by purification using Qragen PCR purification kit, digestion with Ncol followed by incubation in a plate coated with streptavidin Following phenol/CHCl3 purification and ethanol precipitation, the digested DNA was then ligated into a similarly digested pVIII phagemid vector and transformed into ER2738 E cob, then plated on 2% glucose, 2xTY, l00µg/ml ampicillin plates and incubated o/n at 37°C
Individual colonies were picked into 200µl 2% glucose, 2xTY, l00µg/ml ampicillin medium in 96 well plates, and grown at 37°C/200rpm for 6 hours l00µl was transferred to a deep-well plate containing l00µl 2% glucose, 2xTY, 100µg/ml ampicillin plus l0µl M13K07 helper phage/well and incubated for 1 hour without shalcing at 37°C. 500µl per well of 2xTY, l00µg/ml ampicillin/ 25µg/ml kanamycin/ 20µM IPTG medium was added and incubation carried out o/n at 37°C/200rpm
ELISA screening
Round bottom 96 well plates were blocked with 4% Marvel in TBS/0 1% Tween 20 in PBS for I hour at room temperatuxe Picked phage cultures were centrifuged at 3000g for 5 minutes and the phage supernatant was assayed in an ELISA In each well 50µl of phage supernatant were mixed with 5µl Bg spores in
50µl 4% bovine seruin albumin, 1% gelatin m TBS and incubated whilst shaking at loom temperature for 1 hour. The wells were Avashed 5 with 200µl TBS/0.1% Tween20 by centrifugation at 3000g for 5 minutes in between each wash before incubation with anti-M13 horseradish peroxidase conjugated antibody 0 2µg/ml in 4% bovine serum albumin, 1% gelatin in TBS The spores were incubated at room temperature for 1 hour whilst shaking The wells were then washed 5 x with TBS/0 1% Tween20 and the spores were transferred into a fresh plate The spores were then washed once with TBS as described above before development with TMB substrate. The development was stopped with 0.5M H2SO4 and the solution was transfened to a fresh flat-bottomed plate for reading at 450nm Binding data for selected peptides is shown in Figure 7
Example 5. CIS display Library construction, selection & screening against anti-V5 antibody
Library Construction was carried out as described m Example 4.
Round 1 Selection
1x 200µl in vitro transcription/translation reaction (ITT) reaction was set up and incubated at loom temperature for 1 hour as described in Example 4. 1ml of blocking buffer was added to each reaction (Block buffer is 5% skimmed milk powder, l00g/ml sheared salmon sperm DNA, 2.5mg/ml heparimn, m TBS), then placed on ice
for the first round of library selection a 70x11 mm NUNC Maxisorp hnmunotube (Life Technologies, Paisley, Scotland U.K.) was coated with 1ml of 10µg/ml of pol)'clonal anti-V5 peptide antibody (Haiian-Seralab) m PBS for 1 hour at 37°C. The tube was rinsed three times with PBS (fill & empty) and blocked with 3 ml block buffer for 1 hour at 37°C and washed as before Library protein-DNA complexes in block buffer were added, and incubated for 1 hour standing at room temperature. The tube was washed five times with PBS/0 1% Tween 20, then a further five times with PBS only
DNA was eluted into 500µl 1M Sodium acetate pH 5.2 for 10 mmutes on the blood mixer, neutralized with 100µl 1M Tris-HCl pH S.O, then phenol/CHC13 extracted for 5mmutes at 16,100g. DNA was precipitated with 20µg carrier glycogen, ½ volume 7.5M ammonium acetate, and tlrree volumes of ethanol DNA was pelleted at 16,100g for 20 minutes and the pellet washed with 0.5ml 70% ethanol for 5 minutes at 16,100g then vacuum dried, and re-suspended m 20µl water
10µl recovered DNA was PCR amplified m 1x 50µl reaction with primers N0T1RECREV2 (SEQ ID 32) and TACFAR4 (SEQ ID 30) and 2 units of 20.1 Taq DNA polymerase Deep Vent DNA polymerase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds, 72°C, 90 seconds; followed by a 5 minutes extension at 72°C 50µl of reaction product were electrophoresed on a 1% agarose/TAE gel and photographed, then GeneClean purified into 10µl water DNA was reattached to RepA DNA and leamplified for round two as described m example 4 using TACFAR5 (SEQ ID 31) and ORIREV (SEQ ID 02) primers.
Second round selection was carried out as for round 1, using the same primer pairs as described in example 4, with the following changes. Anti-V5 antibody coating concentration was reduced to 5µg/ml Input DNA was approximately 4µg. Third round selection was carried out as for round 2, with the following changes: Approximately 4µg of input DNA was used Recovery PCRs used TACFAR5.1 (SEQ ID 33) and N0TRECREV2 (SEQ ID 32) primers. Finally, pull through PCR used TACFAR6 (SEQ ID 35) and ORIREV (SEQ ID 02) primers for 10 cycles. Round 4 was earned out as for round 3
For cloning out as NcoI-NotI fragments, the stored the lecovered DNA from round 4 the recovered DNA from round 4 was PCR amplified with biotinylated (SEQ ID 26) TAC6 and N0TIREPRECREV2 (SEQ ID 32) primers and cloned into pVIII phagemid vector and electroporated into electrocompetent TG-1 E.coli, as described in example 4
Individual colonies were picked into 200µl 2% glucose, 2xTY, 100µg/ml ampicillin medium in 96 well plates, and grown at 37°C/200rpm for 6 hours. l00µl was transferred to a deep-well plate containing l00µl 2% glucose, 2xTY, 100µg/ml ampicillin plus 109 ku MI3K07 helper phage/well and incubated for 1 hour without
shaking at 37°C. 400^x1 per well of 2xTY, l00µg/ml ampiciliin/ 25µl/ml kanamycin/ 20µM IPTG medium was added and phage amplification continued for 16 hours at 37°C while shaking at 200rpm Bacterial cultures were spun m microtitie plate carriers at 2000g for 10 minutes at 4°C in a benchtop centrifuge to pellet bacteria and culture supernatant used for ELISA
A NUNC Maxisorp ELISA plate was coated with. 100µg/well anti-V5 peptide antibody in l00µl /well PBS for one hour at 37°C The plate was washed 2x200Hl/well PBS and blocked for 1 hour at 37°C with 200µl/well 2% BSA/PBS and then washed 2x200µl/well PBS. 50µl phage culture supernatant was added to each well containing 50µl/well 4% BSA/PBS, and allowed to bind for I hour at room tempeiature The plate was washed two times with 200µl/well PBS/0.1% Tween 20, then two times with 200µl/well PBS Bound phage were detected with 100µl/well, 1.5000 diluted anti-M13-HRP conjugate (Amersham-Pharmacia) m 2% BSA/PBS for 1 hour at room temperature and the plate washed four times as above. The plate was developed for 5 minutes at room temperature with 100µ1/well TMB (3,3',5,5'-Teti-amethylbenzidine) substrate buffer The reaction was stopped with 1 00µl/well 0.5N H2SO4 and read at 450nin Phagemid DNA of ELISA positive clones were then sequenced with standard pUC forward and reverse sequencing primers The amino acid sequence of these clones isolated is shown below Four ELISA positive clones were grown in 10ml culture volumes and phage particles precipitated with PEG-NaCI and re-suspended in 1ml PBS and 50µI retested in ELISA as described above. OD450nm signals against anti-V5 and control anti-ACTH peptide antibody are shown in Figure 8.
Peptide sequences isolated aftei selection
P1C12 CSEQ ID 36) CGCPTMAARVRPVLNSKH
P2H1 (SEQ ID 37) MTTVPVLMISV
P1B5 (SEQ ID 38) TLSTRHHNVIDRFNLRNF
P2BS (SEQ ID 39) SIRTLTGSTPAQFDATAD
Example 6. Selection of ovalbutmin binding peptides from a CIS display library'
For any selection methodology it is important that the selected entities are capable of binding to the target selected against, independently of the carrier molecule associated with it during selection and screening. In this example, selected peptides are selected and synthesized to allow confirmation of target binding. Random 12mer peptide library construction was carried out as described in Example 3 Four rounds of selection were carried out as described in example 4 with 100µg/ml ovalbumin (SIGMA, Dorset, UK) coated onto immunotubes.
For clomng out as NcoI-NotI fragments, the recovered DNA from round 4 was PCR amplified with biotmylated TAC6 (SEQ ID 26) and N0TIREPRECREV2 (SEQ ID 32) primers and cloned into a pVIII phagemid vector and electroporated into electiocoinpetent TG-1 E coli, as described in example 4
Individual colonies were picked into 200µl 2% glucose, 2xTY, l00µg/ml ampicillin medium in 96 well plates, and grown at 37°C/200rpm for 6 hours 100µl was transferred to a deep-well plate containing l00µl 2% glucose, 2xTY, 100µg/ml ampicillin plus 109 kr'u M13K07 helper phage/well and incubated for 1 hour without shaking at 37°C 400µl per well of 2xTY, l00µg/ml ampicillin/ 25µg/ml kanamycin/ 20pM IPTG medium was added and phage amplification continued for 16 hours at 37°C while shaking at 200rpm Bacterial cultures were spun m microtitre plate carriers at 2000g for 10 minutes at 4°C in a benchtop centrifuge to pellet bacteria and culture supernatant used for ELISA
A NUNC Maxisorp ELISA plate was coated with 100µg/well ovalbumin in 100µl /well PBS overnight at 4°C The plate was washed 2x200µl/well PBS and blocked for 1 hour- at 37°C with 200µl/well 2% BSA/PBS and then washed 2x200µl/well PBS 50µl phage culture supernatant was added to each well containing 50µl/well 4% BSA/PBS, and allowed0 to bind for 1 hour at room temperature The plate was washed two times with 200µl/well PBS/0 1% Tween 20, then two times with 200µl/well PBS Bound phage were detected with 100µl/well, 1 '5000 diluted anti-M13-HRP conjugate (Amersham-Pharmacia) m 2% BSA/PBS for 1 hour at room temperature and the plate washed four times as above The plate was developed for 5 minutes at room temperature with l00µl/well TMB (3,3\5,5'-
Tetramethylbenzidine) substrate buffer The reaction was stopped with 100µl/well 0.5N H2SO4 and read at 450nm. Phagemid DNA of ELISA positive clones were then sequenced with M13REV primer. The ammo acid sequence of these clones isolated is shown below
CI(SEQID40) ANLWRIVLHGWW
C4(SEQID41) VSFMLLGPHRHR
Co (SEQ ID 42) L V L H W L S L G S R
C8 (SEQ ID 43) SNQVVLILHLRP
Contiol(SEQID44) AESWLHQSWIHL
Peptide sequences from foui lepresentative ELISA positive clones were synthesized (SIGMA-Genosys Ltd) with brotin added to the C-tenmnus to and detection in ELISA These peptides weie tested in ELISA against ovalbumin, along with a control peptide previously isolated by phage display selection against B globigii spores. A NLINC Maxisorp ELISA plate was coated with l00µg/well ovalbumin in 100µ1 /well PBS, or, 200ng/well ant-V5 polyclonal antibody in PBS, overnight at 4°C. The plate was washed 2x200µl/well PBS and blocked for 1 hour at 37°C with 200µl/well 2% skimmed milk powder/PBS and then washed 2x200µl/well PBS. lµg of diluted peptides were added to each well in l00µl/well 2% BSA/PBS, and allowed to bind for 1 hour at 100m tempeiature The plate was washed two times with 200µl/well PBS/0.1% Tween 20, then two times with 200µ1/well PBS Bound peptides were detected with 100µl/well, 1 2000 diluted streptavidin-HRP conjugate (Pierce) m 2% BS A/PBS for 1 hour at room temperature and the plate washed four times as above The plate was developed for 5 minutes at room temperature with l00µl/well TMB (3,3',5,5'-Tetiamethylbenzidine) substrate buffer. The reaction was stopped with 100µl/well 0.5N H2SO4 and lead at 450nm (Figure 9)
Example 7. Display of single-chain Fv antibody (scFv) fragments in a CIS display system
A tac-scFv-RepA-CIS-on construct was constructed by PCR overlap extension essentially as described previousl}' in example 1, Anti-mecoprop scFv DNA (Haptogen Ltd, Aberdeen, UK) was amplified in a 50µl reaction volume containing 200|iM dNTPs, IxNEB polymerase amplification buffer (l0mM KCl, l0mM (NH4)2S04, 20mM Tris-HCl pH 8 S, 2mM MgS04, 0 1% TritonX-l00) with lOpmol of each of the primers TACMECOFOR (SEQ ID 45) and REPAMECOBAK (SEQ ID 46) and 2 units of 20 1 Taq DNA polymerase. Deep Vent DNA polymerase mixture (NEB) foi 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds; 72°C, 80 seconds; followed by a 5 minutes extension at 72°C. Products were electrophoiesed on a 1% agarose/TAE gel and purified with a Geneclean II kit into 20µl water. This was assembled with RepA-CIS-ori DNA generated with ORIREV408 (SEQ ID 20) and MECOREPAFOR (SEQ ID 47), and Tac promoter DNA generated with TACFARUP (SEQ ID 23) and MECOTACBAK (SEQ ID 48) in a 50µl reaction volume containing 200µM dNTPs, IxNEB polymerase amplification buffer (l0md KCl, l0M (NH4)2S04, 20mM Tris-HCl pH 8.8, 2mM MgS04, 0.1% TritonX-l00) with l0pmol of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) and 2 units of 20:1 Taq DNA polymerase. Deep Vent DNA polymerase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds, 72°C, 80 seconds, followed by a 5 minutes extension at 72°C. Products weie electrophoresed on a 1% agarose/TAE gel and purified with a Geneclean II kit mto 20µl water.
DNA was reamplified in l0x 50µl reactions containing 200µM dNTPs, IxNEB polymerase amplification buffer (l0mM KCl, l0mM (NH4)2S04, 20mM Tris-HCl pH 8 8, 2mM MgS04, 0 1% TritonX-100) with l0pmol of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) and 2 units of 20.1 Taq DNA polymerase. Deep Vent DNA polymerase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds; 72°C, 80 seconds, followed by a 5 minutes extension at 72°C Products were electrophoiesed on a 1% agarose/TAE gel and purified with a Geneclean II kit mto 100µl water
ScFvDNA was then translated in the following two reaction conditions:
(Table Removed)
Reactions were incubated at 30°C for 30 minutes then 1 µl 0.25M ox-glutathione added to reaction 2 and incubation at 30°C continued for a further 30 minutes 1ml of blocking buffei was added to each leaction (Block buffer is 1% gelatin, 100µg/ml sheared salmon sperm DNA, 2 5mg/ml heparin, in TBS), spun at 10,000g for 2 minutes, then placed on ice.
NUNC star immunotubes were coated with 0 5ml 10 µg/ml BSA-mecoprop conjugate, or 10µg/ml BSA in PBS for 1 hour at 37°C. Tubes were washed 2x PBS. then blocked for 1 hour at room temperature with 3ml blocking buffer on a blood mixer, then tubes were washed 2x PBS
0.5ml of each diluted ITT was added to either a blocked BSA coated or BSA-mecoprop coated tube and incubated at loom temperature for 1 hour. Tubes were washed 5x TBS/0 1% Tween 20, 5x TBS
Bound DNA was eluted for 10 minutes at room temperature with 0.5ml of 0.5M NaCI/l0mM Tris pH 8, ImM EDTA, then extracted with 0 5mL phenol/chloroform and precipitated with 20µg carrier glycogen,½: volume 7.5M anmomum acetate, and three volumes of ethanol DNA was pelleted at 14,000g for 20 minutes and the pellet washed with 0 5ml 70% ethanol for 5 minutes at 14,000g then vacuum dried, and re-suspended in 20µl.I water.
l0 µl of recovered DNA was PCR amplified in a 50µl leaction volume containing 200µM dNTPs, IxNEB polymeiase amplification buffer (lmM KCl, l0mM (NH4)2S04, 20mM Tris-HCl pH S 8, 2mM MgS04, 0 1% TritonX-100) with lOpmol of each of the primers TACMECOFOR (SEQ ID 45) and REPAMECOBAK (SEQ ID 46) and 2 units of 20.1 Taq DNA polymerase. Deep Vent DNA polymeiase mixture (NEB) for 30 cycles of 94°C, 40 seconds, 60°C, 40 seconds, 72°C, 80
seconds; followed by a 5 minutes extension at 72°C Products were electrophoresed on a 1 % agarose/TAE gel and photographed. Greater amounts of DNA were observed from selections on antibody target than with recovered from BSA coated tubes, indicating that functional scFv-RepA-DNA complexes were being selected (Figure 10).




WE CLAIM:
1. A method for producing an in-vitro peptide expression library
characterized in that each peptide of said expression library is non-
covalently linked to a DNA construct that includes a sequence that encodes
that peptide,
the method comprising the steps of:
(a) providing a DNA construct comprising:
(i) a DNA sequence of the kind such as herein described,
capable of being bound by the peptide defined in (iii);
(ii) DNA encoding a peptide; and
(iii) DNA encoding a peptide capable of non-covalently binding
directly or indirectly to said DNA sequence of (i); wherein the
DNA construct and the protein that it encodes are selected to
have cis-activity;
(b) expressing more than one DNA constructs according to (a) in
vitro wherein the DNA constructs encode more than one
different peptides such that each expressed peptide is non-
covalently linked to the DNA construct from which it was
produced.
2. A method as claimed in claim 1 wherein the DNA construct of
(a) optionally comprises:
(iv) a DNA element that directs cis-activity.
3. A method as claimed in claim 1 or 2, wherein the peptide
encoded by the DNA of (iii) is capable of non-covalently binding, directly to
the DNA sequence of (i).
4. A method as claimed in claim 3, wherein the DNA of(ii) is linked to the DNA of(i) and (iii) by restriction enzyme digestion and ligation.
5. A method as claimed in any one of claims 2 to 4, wherein the DNA construct of(a) optionally comprises:

(v) DNA encoding a fragment of a repA protein, wherein the
fragment comprises at least the C-terminal 20 amino
acids of a repA protein, and wherein the fragment is
capable of interacting with the DNA element of (iv);
and wherein the DNA element of(iv) is located 3' to the DNA of(ii), (iii)
and (v) in the DNA construct.
6. A method as claimed in claim 5, wherein the peptide encoded by the DNA of (iii) is a repA protein and wherein the DNA sequence of (i) is ori
7. A method as claimed in claim 5 or 5, wherein the repA protein is selected from repA of the IncI complex plasmids and repA of the IncF, IncB, IncK, IncZ and IncL/M plasmids.
8. A method as claimed in claim 6, wherein said DNA construct comprises the sequence encoding a repA protein, the cis DNA element and the ori DNA of the IncFII plasmid Ri.
9. A method as claimed in any one of claims 5 to 8, wherein the repA protein has the sequence given in SEQ ID NO: 16 and wherein the DNA element of (iv) has the sequence given in SEQ ID NO: 17.
10. A method as claimed in any one of the preceding claims wherein DNA of the DNA construct that is not bound by the peptide encoded by the DNA of (iii) is bound by non-specific DNA binding protein.
11. A method as claimed in claim 4, wherein the peptide encoded by the DNA of (iii) is an oestrogen receptor DNA binding domain and wherein the DNA sequence of (i) is an oestrogen receptor target sequence.
12. A method as claimed in claim 11, wherein the oestrogen receptor DNA binding domain comprises amino acids 176 to 282 of the oestrogen receptor DNA binding fragment and wherein the DNA sequence

if(i) comprises the oestrogen receptor target sequence given in SEQ ID NO: 14.
13. A method as claimed in any one of the preceding claims wherein
the DNA is under the control of suitable promoter and translation sequences
of the kind such as herein described to allow for in vitro transcription and
translation.
14. A method as claimed in any one of the preceding claims
wherein the peptide of (ii) is an antibody or fragment thereof.
15. A method as claimed in any one of the preceding claims wherein said method produces a mixture of at least 104 molecules.
16. A method as claimed in any one of the preceding claims wherein step (b) of the method is optionally carried out in the presence of a compound of the kind such as herein described that prevents nuclease activity, or reduces non-specific DNA-protein or protein-protein interactions.
17. A method as claimed in any one of the preceding claims wherein step (b) of the method is carried out in a coupled bacterial transcription/ translation environment.
18. A method as claimed in claim 17, wherein the coupled bacterial
transcription/ translation environment is the S30 extract system.
19. An in-vitro peptide expression library produced according to the
method of any one of claims 1 to 18.

20. A DNA construct as described in any one of claims 1 to 18

Documents:

634-delnp-2005-abstract.pdf

634-DELNP-2005-Claims.pdf

634-delnp-2005-complete specification (as,files).pdf

634-delnp-2005-complete specification (granted).pdf

634-delnp-2005-Correspondence-Others (05-11-2009).pdf

634-delnp-2005-correspondence-others.pdf

634-delnp-2005-correspondence-po.pdf

634-DELNP-2005-Description (Complete).pdf

634-delnp-2005-form-1.pdf

634-delnp-2005-form-18.pdf

634-DELNP-2005-Form-2.pdf

634-delnp-2005-Form-3 (05-11-2009).pdf

634-delnp-2005-form-3.pdf

634-delnp-2005-form-5.pdf

634-delnp-2005-gpa.pdf

634-delnp-2005-pct-101.pdf

634-delnp-2005-pct-210.pdf

634-delnp-2005-pct-304.pdf

634-delnp-2005-pct-308.pdf

634-delnp-2005-petition-137.pdf


Patent Number 242766
Indian Patent Application Number 634/DELNP/2005
PG Journal Number 38/2010
Publication Date 17-Sep-2010
Grant Date 09-Sep-2010
Date of Filing 17-Feb-2005
Name of Patentee ISOGENICA LIMITED,
Applicant Address STUART HOUSE CITY ROAD, PETERBOROUGH PE1 1QF, ENGLAND,
Inventors:
# Inventor's Name Inventor's Address
1 PHILIP KUHLMAN 3 LAWRY CLOSE ST LVES. CAMBRIDGESHIRE PE27 3EF,ENGLAND
2 DAVID COOMBER 6 WARRIGAL PLACE, ORANGE, NSW 2800, AUSTRALIA
3 DUNCAN MCGREGOR, 21 HOOPER STREET, CAMBRIDGE CB1 2NZ, ENGLAND
4 RICHARD ODEGRIP HANTVERKARGATAN 60, 112 31 STOCKHOLM,SWEDEN
5 KEVIN FIZGERALD THE COBB HIGH STREET, HEMPSTEAD, EXXEX CB10 2PE,ENGLAND
6 ROSEMARIE HEDERER 1C MORTLOCK AVENUE, CAMBRIDGE CB4 1TD, ENGLAND
7 BILL ELDRIDGE 19 LITTLE WALDEN ROAD, SAFFRON WALDON, ESSEX, CB10 2DT, ENGLAND
8 CHRIS ULLMAN 10 CRANLEIGH CLOSE, CAMBRIDGE CB2 2NP, ENGLAND
PCT International Classification Number C12N 15/00
PCT International Application Number PCT/GB2003/003860
PCT International Filing date 2003-09-05
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 0220759.5 2002-09-06 U.K.
2 0304521.8 2003-02-27 U.K.
3 0304657.0 2003-02-28 U.K.