Title of Invention

METHOD AND DNA VECTOR FOR PRODUCING A RECOMBINANT PROTEIN IN YEAST CELLS

Abstract Methods and compositions are provided relating to production of recombinant protein in yeast. A modified PLAC4 is described where one or more mutations may be introduced into the Pribnow box-like sequences in the promoter. The modified promoter when placed upstream of a target gene in a vector causes a significant reduction of target gene expression in transformed bacteria but produces efficient expression of the target gene in yeast.
Full Text FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
'Method for construction and use of Kluyveromyces Lactis
Promoter Variants in K. Lactis that substantially lack
E. Coli Transcriptional capability"
NEW ENGLAND BIOLABS, INC., of 240 County Road, Ipswich, MA 01938, USA
The following specification (particularly) describes the nature of the invention and the manner in which it is to be performed

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Method for Construction and Use of Kluyveromyces lactis Promoter
Variants in K. lactis that Substantially Lack
E. coli Transcriptional Capability
5 BACKGROUND
For over a decade, the budding yeast Kluyveromyces lactis (K. lactis) has been widely used for industrial-scale production of recombinant proteins in the food and dairy industries for reasons
10 that include the following factors: (i) many strains of K. lactis grow rapidly and to extremely high cell densities in culture; (ii) K. lactis efficiently directs proteins to be secreted into the medium; and (iii) K. lactis has GRAS (Generally Regarded As Safe) FDA status which permits its use for food, agricultural and health-related applications.
15
A typical K. lactis heterologous protein production strategy involves directing a desired protein to be secreted from the cell into the growth medium. This methodology has a number of advantages over cellular expression methods: (i) the protein is
20 produced significantly pure since K. lactis secretes relatively few endogenous proteins; (ii) post-translational protein modifications found only on secreted eukaryotic proteins are obtainable; and (iii) strategies to harvest protein from the medium of continuously growing cells can be devised.
25
A strong yeast promoter suitable for directing high levels of transcription in K. lactis is the K. lactis LAC4 promoter (PLAC4 (Dickson, et al. Cell 15:123-130 (1978); Dickson, R. C, and M. I. Riley, Biotechnology 13:19-40 (1989); Dickson, et al. Mol. Cel. Biol.
30 1:1048-1056 (1981)). This promoter naturally drives expression of the LAC4 gene which encodes a highly expressed lactase (

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galactosidase). Transcription of LAC4 is elevated in response to the presence of lactose or galactose in growth medium where lactase allows the cell to convert lactose to fermentable sugars. Expression of heterologous proteins from Puc4 may achieve levels greater than 100 mg L"1 of secreted recombinant protein in yeast fermentations.
Unfortunately, in addition to its ability to function as a strong promoter in K. lactis, PLAC4, constitutively promotes gene expression in E. coli cells. This can be particularly problematic when trying to assemble DNA constructs harboring genes that encode a protein toxic to E. coli prior to their introduction into yeast cells. One approach to solving this problem has been reported by Gibbs et al. {FEMS Yeast Research 4: 573-577 (2004)) who utilized yeast introns in the shuttle vector. Unfortunately, this modification abolishes some but not all functional expression of potentially toxic recombinant proteins.
SUMMARY
20 In an embodiment of the invention, a method is provided for producing a recombinant protein in yeast cells that includes the steps of: obtaining a vector into which a gene encoding the target protein has been inserted together with a modified PLAC4 wherein the modification results in a significant reduction in gene expression in
25 bacteria exemplified by E. coli; transforming yeast cells exemplified by K. lactis with the vector; and producing an effective amount of recombinant protein in the yeast cells. In certain embodiments, at least 50%, more particularly at least 70%, more particularly at least 90%, of the transformed yeast cells express recombinant protein.
30 In an embodiment of the invention, the effective amount of

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recombinant protein produced in yeast is substantially similar to the amount of protein from a recombinant gene under control of an unmodified Puc4 promoter.
5 The modified PLAC4 in the method may optionally include a
mutation in one or more Pribnow box-like sequences, for example in PBI, PBII and PBIII, more particularly in a first region of the promoter corresponding to nucleotides -198 to -212 or in a second region of the promoter corresponding to nucleotides -133 to -146.
10 In certain embodiments, the modified PLAC4 contains one or more mutations in both the first region and also one or more mutations in the second region of the promoter. In a further embodiment of the invention, nucleotides -1 to -283 in the modified PLAC4 are substituted by nucleotides -1 to -283 of the phosphoglycerate
15 kinase promoter from S. cerevisiae (PGK1).
The vector may be an episomal or an integrative plasmid in the transformed yeast cells. The vector contains a modified PLAC4 promoter and optionally a PLAC4 terminator. Moreover, the vector
20 may include a DNA sequence encoding at least one of a yeast
secretion signal peptide such as K. lactis a-mating factor (Kl a-MF), a selectable marker such as Aspergillus nidulans acetamidase (amdS) selectable marker gene, or a multiple cloning site for insertion of a gene encoding a recombinant protein.
25
The cells transformed with the above-described vector may include a host yeast cell and /or a host bacterial cell.
In an embodiment of the invention, a kit that includes a 30 vector as described above and optionally includes competent yeast

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cells together with instructions for use is provided.
An embodiment of the invention provides a modified PLAC4 Pribnow box wherein TTATCATTGT (SEQ ID NO:22) is modified to
5 AGAACAGAGA (SEQ ID NO:23) and/or TATTATTCT is modified to GAGAGCTCT.
DESCRIPTION OF FIGURES
10 Figure 1 shows the E. coli/K. lactis integrative expression
vector pGBNl.
Genes are cloned into the multiple cloning site (MCS) in the same translational reading frame as the S. cerevisiae a-mating
15 factor secretion leader sequence (Sc a-MF). Transcription is initiated and terminated by PLAC4 and LAC4 transcription terminator sequence (TTLACA), respectively. The S. cerevisiae ADH1 promoter (PADHI) drives expression of a bacterial gene conferring resistance to G418 in yeast. E. coli vector sequence has been inserted into a unique SacII
20 site in PLAC4, to allow for propagation in E. coli. The vector is
linearized by digestion with SacII OR BstXI for integration into the LAC4 promoter locus in the K. lactis chromosome.
Figure 2 shows the Pribnow box-like sequences in PLAC4 and
25 construction of PLAC4 variant expression vectors.
Figure 2A shows Pribnow box-like sequences PBI, and PBII and PBIII (SEQ ID NOS:l and 2) relative to the major and minor E. coli transcription start sites associated with PLAC4, and are aligned
30 with the Pribnow box consensus sequence TATAAT. Nucleotides that

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agree with the consensus sequence are boxed.
Figure 2B shows expression vectors containing PLAC4 variants. The approximate positions of the E. coli major and minor
5 transcription start sites are shown in the schematic for pGBNl. The approximate positions of the galactose-responsive elements, upstream activator sequence (UAS) UASI and II, are shown for each construct. Regions of PLAC4 DNA that have been replaced with fragments of the PGK1 promoter are shown in black. Mutated bases
10 in the Pribnow box-like sequences in the PLAC4 DNA of plasmids pGBNlpB1 and pGBN1PBII-PBIII are indicated with a black dot above each base (SEQ ID NOS:3 and 4). All numbered positions are relative to the adenine of the ATG start codon of the Sc a-MF secretion leader that has been designated position +1.
15
Figure 3 shows PLAC4 variant expression of green fluorescent protein (GFP) in E. coli and human serum albumin (HSA) in K. lactis.
20 Figure 3A shows GFP cloned downstream of each of the
various PLAC4 variants. Proteins from lysates of E. coli carrying each expression construct were separated by SDS-PAGE, and GFP was detected by Western analysis.
25 Lane 1: pGBNl used as a negative control. Lysate is derived
from bacteria containing an empty pGBNl plasmid;
Lane 2: pGBNl/P^^ used as a second control containing an unmodified PLAC4
30

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Lanes 3-6: lysates used from E. coli transformed with pGBNl in which P^ has been substituted with PPGK1, PHybrid, PLAC4-PBI and
PLAC4-PBII-PBIII
5 Figure 3B shows HSA cloned downstream of each PLAC4, for
expression in K. lactis cells. Secreted proteins in spent culture medium of K. lactis strains containing the various integrated HSA expression vectors were resolved by SDS-PAGE (4-20% acrylamide) and Coomassie stained. HSA ran as a single band with an apparent
10 mass of 66 kDa.
Lane 1: spent culture medium from a yeast strain containing empty pGBNl integrated into the chromosome as a negative control; 15
Lanes 2-6: spent media from K. lactis transformed with pGBNILAC4-HSA/PLac4, pGBNIPGKI -HSA/PPGK1, pGBNIHybrid-HSA/PHybrid, PGBNILAC4-PBI-HSA/PLAC4-PBI and PGBNILAC4-PBII-PBIII-HSA/PLAC4-PBII-PBIII.
20 Figure 4 shows pKLACl, an E. coli/K. lactis integrative
expression vector. The pKLACl vector (GenBank No. AY968582) is organized similarly to pGBNl with the following modifications: (i) genes are cloned into the multiple cloning site in the same translational reading frame as the native Kl a-MF leader sequence;
25 and (ii) expression in K. lactis is initiated by the PLAC4.PBIi variant. The PADHI drives expression of an acetamidase-selectable marker (amdS) gene for selection of transformants by growth on acetamide medium.
30 Figure 5 shows the activity of secreted enterokinase in the

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spent culture medium of K. lactis cells containing integrated pKLACl-EKL(the gene encoding the enterokinase catalytic subunit). Seven K. lactis strains harboring pKLACl-EKL and wild-type GG799 cells were grown in YPGal medium for 48 hours. Cleared spent
5 culture medium was assayed for enterokinase activity by measuring cleavage of a fluorogenic peptide over time. KLEK-S1 and KLEK-S4 are two strains that contain multiple copies of integrated pKLACl-EKL as determined by Southern analysis. All other strains contain a single integrated copy of PKLACI-EKL.
10
DETAILED DESCRIPTION
A functional shuttle vector allows for the propagation of cloned genes in bacteria prior to their introduction into yeast cells
15 for expression. However, yeast expression systems that utilize the strong PLAC4can be adversely affected by the serendipitous expression of protein from genes under control of PLAC4, in bacterial host cells such as E. coli. This promoter activity can interfere with the cloning efficiency of genes whose translational products are
20 potentially detrimental to bacteria.
Two nucleotide sequences in the PLAC4, closely resemble the bacterial Pribnow box transcription element consensus sequence, which is TATAAT. These sequences are located approximately 10
25 nucleotides upstream from the site where transcription begins and are adjacent and upstream of the major and a minor transcription start sites in E. coli (Dickson et al. Biotechnology 13:19-40 (1989)). In particular, the sequences are located at -204 to -209 for the major transcript, and -144 to -136 for the minor transcript) (see
30 boxed sequences in Figure 2A).

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The initiation sites of two RNA transcripts associated with E. coli expression of K. lactis P^^ have been previously mapped to -196 bp (initiation of the major E. coli transcript) and -127 bp 5 (initiation of the minor E. coli transcript) relative to the adenine nucleotide in the ATG start codon of the native LAC4 gene (Dickson et al. 1989).
PLAC4 variants with mutated Pribnow box-like sequences can
10 be created by site-directed mutagenesis which substantially retain their ability to function as strong promoters in K. lactis to the extent similar to that of unmutated Pribnow box-like sequences. Pi_ac4 variants that have mutated Pribnow box-like sequences may retain strong promoter activity in other yeast strains from the
15 Kluyeromyces species as well as Saccharomyces species, Pichia species, Hansenula species, Yarrowia species, Neurospora species, Aspergillus species, Penicillium species, Candida species, Schizosaccharomyces species, Cryptococcus species, Coprinus species, Ustilago species, Magnaporth species and Trichoderma
20 species. Based on the knowledge in the art that DNA sequence is determinative for promoter strength, it is expected that some mutants will produce greater amounts of protein than under similar conditions using the wild-type PLAC4,. Mutation is here intended to mean any of: a substitution, a deletion or an addition of one or
25 more nucleotides in a DNA sequence.
In an embodiment of the invention, the fungal expression host is the yeast K. lactis and the bacterial host is E. coli and a series of PLAC4 variants have been created by targeted mutagenesis 30 of three DNA sequences (PBI, PBII and PBIII) that resemble the E.

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coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P^. In the examples, the mutation in PLAC4, is in the region of (a) the -198 to -212 region of the promoter (Figure 2B)
5 for example at positions -201, -203, -204, -207, -209 and -210. These mutations do not substantially interfere with the ability of the promoter to function as a strong promoter in K. lactis; (b) the -133 to -146 region of the promoter for example at positions -139, -140, -141, -142 and -144 which do not substantially interfere with strong
10 promoter activity; or (c) the -198 to -212 and -133 to -146 regions. In a further embodiment, a hybrid promoter was created that consists of 283 bp (-1 to -283) of the S. cerevisiae (Sc) PGKI promoter replacing the -1 to -283 region of K. lactisPLAC4 (Figure 2B).
15
Overexpression of proteins in K. lactis and more generally in yeast involves construction of a shuttle vector containing a DNA fragment with sequences suitable for directing high-level transcription of a gene of interest upon introduction into the yeast
20 host. The vector should contain at least one or more of the following: (i) a strong yeast promoter; (ii) DNA encoding a secretion leader sequence (if secretion of the protein into the medium is desired); (iii) the gene encoding the protein to be expressed; (iv) a transcription terminator sequence; and (v) a
25 yeast-selectable marker gene. These sequence components are typically assembled in a plasmid vector in E. coli then transferred to yeast cells to achieve protein production.
PLAC4 can function as a strong promoter for protein expression 30 in yeast when present on an integrative plasmid or an episomal

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plasmid such as pKDl-based vectors, 2 micron-containing vectors, and centromeric vectors. The secretion leader sequence (if secretion of the protein into the medium is desired) may include a Sc a-MF pre-pro secretion leader peptide which has been cloned as a
5 Hindlll/Xhol fragment. Other prokaryotic or eukaryotic secretion signal peptides (e.g. K. lactis a-mating factor pre-pro secretion signal peptide, K. lactis killer toxin signal peptide) or synthetic secretion signal peptides can also be used. Alternatively, a secretion leader can be omitted from the vector altogether to
10 achieve cellular expression of the desired protein.
An example of a transcription terminator sequence is TTLAC4.
The yeast-selectable marker gene can be for example, G418
15 or an amdS gene. Expression of acetamidase in transformed yeast cells allows for their growth on medium lacking a simple nitrogen source but containing acetamide. Acetamidase breaks down acetamide to ammonia which can be utilized by cells as a source of nitrogen. A benefit of this selection method is that it enriches
20 transformant populations for cells that have incorporated multiple tandem integrations of a pKLACl-based expression vector and that produce more recombinant protein than single integrations (Figure 5).
25 The above-described mutants P^ have been integrated into
an E. coli/K. lactis integrative shuttle vector, for example, pGBNl and pKLACl shown in Figures 1 and 4, respectively, which integrates into the K. lactis genome after transformation of competent host cells and subsequently directs protein expression.

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In embodiments of the invention, at least 50%, more specifically at least 70%, preferably at least 90%, of transformants that form on acetamide plates following transformation of K. lactis with pKLACl-based constructs express foreign protein, for example,
5 HSA or the E. coli maltose-binding protein (MBP), toxic protease enterokinase, mouse transthyretin, toxic glue proteins from marine organisms and a bacterial cellulase. These examples are not intended to be limiting. The system has utility for any protein-encoding gene placed downstream of the mutated P^^.
10
Levels of protein expression under PLAC4, and mutants thereof were determined for several different proteins. For example, mutation of PBI reduced bacterial expression of a reporter protein (GFP) by ~87%, whereas mutation of PBII and PBIII had little effect
15 on GFP expression in the bacterial host cell. Deletion of all three sequences completely eliminated GFP expression in the bacterial host cells. For HSA, the Example and Figure 3b show that about 50 mg L"1 of HSA was secreted by K. lactis when expressed from either wild-type or mutant PLAC4
20
The references cited above and below as well as U.S. Provisional Application Serial No. 60/560,418 filed April 8, 2004 are herein incorporated by reference.
25 EXAMPLE
Yeast strains, transformation and culturing conditions
The prototrophic K. lactis strain GG799 (MAT a [pGKll+]) 30 was routinely grown and maintained on YPD media (1% yeast

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extract, 2% peptone, 2% glucose) at 30°C. Prior to transformation of GG799 cells, 1 of pGBNl- or pKLACl-based expression vector containing a gene of interest was linearized by SacII digestion. Linearized expression vectors were used for integrative
5 transformation of commercially available competent K. lactis GG799 cells (New England Biolabs, Beverly, MA) as directed by the supplier. Colonies of cells transformed with pGBNl, PGBNIPGKI, pGBNlHyb/ PGBNIPBI or PGBNIPBH-PBIII vectors were selected by growth on YPD agar plates containing 200 \ig G418 ml"1 (Sigma, St.
10 Louis, MO) for 2-3 days at 30°C. Colonies of cells transformed with pKLACl-based vectors were selected by growth on agar plates containing 1.17% yeast carbon base (New England Biolabs, Beverly, MA), 5 mM acetamide (New England Biolabs, Beverly, MA) and 30 mM sodium phosphate buffer pH 7 for 4-5 days at 30°C. K. lactis
15 strains expressing heterologous genes were cultured in YP media containing 2% galactose (YPGal) at 30°C for 48-96 hours.
Polymerase chain reaction
20 Primers used in this study are listed in Table 1. Amplification
by PCR was performed using high fidelity Deep Vent™ DNA polymerase (New England.Biolabs, Beverly, MA). Typical PCR mixtures contained 0.2 mM dNTPs, 0.5 ug of each primer, IX Thermopol buffer (New England Biolabs, MA) and 100 ng template
25 DNA in a total reaction volume of 100 pi. Thermocycling typically consisted of a "hot start" at 95°C for 5 minutes followed by 30 cycles of successive incubations at 94°C for 30 sec, 58°C for 30 sec and 72°C (1 min per kb of DNA). After thermocycling, a final extention was performed at 72°C for 10 minutes.

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Construction of K. lactis PLAC4 variants in pGBNl
All promoter variants were derived from wild-type P^^ present in the integrative expression vector pGBNl, a K. lactis/E.
5 coli shuttle vector that contains 2317 bp of P^ DNA split into 1663 and 654 bp fragments that are separated by pUC19 plasmid DNA (Figure 1). The split occurs at a unique restriction site recognized by SacII. A 2830 bp of pUC19 vector DNA sequence has been inserted at this unique restriction site. This allows the expression vector to
10 integrate into the promoter region of the native LAC4 locus in the K. lactis chromosome after digestion with SacII or BstXI and introduction into yeast cells. Additionally, K. lactis DNA that directs integration of the vector into the K. lactis chromosome at locations other than LAC4 can be inserted into the vector. Any DNA
15 containing a bacterial origin of replication and a selectable marker gene can be used in place of the pUC19 DNA sequence. The position of the wild-type PLAC4 sequence, or any PLAC4, mutant or hybrid cloned into pGBNl is immediately upstream of the coding region for the secretion leader sequence. 20 Additionally, pGBNl contains DNA encoding the Sc a-MF pre-pro domain immediately downstream of P^ to direct secretion of heterologously expressed proteins. Finally, pGBNl carries a geneticin (G418) resistance gene expressed from the PADH, for
25 dominant selection in yeast. To create plasmid pGBNlPGK, a
Pmll/Hindlll fragment containing 488 base pairs of the S. cerevisiae PGK1 promoter was cloned into the Hpal/Hindlll sites of plasmid pGBNl to replace 1067 base pairs of native PLAC4 (Figure 2B). Primer PI and primer P2 were used to amplify 283 base pairs of the S.
30 cerevisiae PGK1 promoter using plasmid pGBNiPGK1 as a template.

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The 283 bp fragment was cloned into the SnaBI/Hindlll sites of plasmid pGBNl to produce plasmid pGBNlHyb. Primer P3 was designed to incorporate mutations into the putative Pribnow boxlike sequence (PBI) that lies upstream of the E. coli major
5 transcription start site as detailed in Figure 2B. Primers P2 and P3 were used to amplify a PLAC4 fragment containing mutations in PBI using plasmid pGBNl as a template. Amplified DNA from this initial PCR was used as template for a" second PCR using primers P2 and P4. The final DNA product was cloned into the SnaBI/Hindlll sites of
10 plasmid pGBNl to produce plasmid pGBNlPB1. A PCR knitting method was used to mutate the PBII and PBIII sequences (Figure 2B) that lie upstream of the E. coli minor transcription start site using complementary primers P5 and P6. Primers P2 and P5 and primers P4 and P6 were used to amplify 586 bp and 160 bp mutated PLAC4
15 DNA fragments, respectively. Each amplified DNA product was
purified by QiaQuick™ PCR purification spin column chromatography (Qiagen, Valencia, CA) and combined as template in a second amplification reaction containing primers P2 and P4. The amplified PLAC4DNA fragment containing mutagenized PBII and PBIII sites was
20 cloned into the SnaBI/Hind III sites of plasmid pGBNl to produce plasmid pGBNlPBII.PBIII.
Targeted mutagenesis of Pribnow box-like sequences in PLAC4
25 A series of four PLAC4 variants were generated to eliminate the
E. coli promoter activity of PLAC4 by either replacing or introducing point mutations in PBI and PBII/PBIII as shown in Figure 2B.
Vector pGBNlpcK, incorporates 485 bp of the S. cerevisiae
30 PGK1 promoter (PPGKI) in place of 106*7 bp of native PPAC4, thereby

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removing both galactose-responsive upstream activating sequences (UASI and UASII) and all three Pribnow box-like sequences.
(ii) Vector pGBNlHyb incorporates 283 bp from the 3' end of P 5 PPGKI in place of 283 bp comprising the 3' end of P^ resulting in deletion of all three Pribnow box-like sequences but leaving both UAS sequences intact.
(iii) Vector pGBNlPB, contains 6-point mutations that eliminate
10 the Pribnow consensus sequence of PBI between nucleotides -204 and -209 of PLAC4.
(iv) Vector pGBNipB„.pBIII contains 5-point mutations that eliminate the Pribnow consensus sequences of PBII and PBIII 15 between nucleotides-136 and-144 of PLAC4-
Cloning and expression analysis of GFP in E. coli
GFP was PCR amplified with primers P7 and P8 using plasmid 20 pGFPuv (Clontech, Palo Alto, CA) as a template. Amplified GFP was cloned in-frame with the a-MF pre-pro domain in the Bglll/NotI sites of the various pGBN vectors (see previous section). Lysates of bacteria containing various pGBN-GFP constructs were prepared from 50 ml overnight cultures grown at 30°C in LB medium
25 containing 100 ug/ml ampicillin. Cultures were centrifuged and the cell pellets were frozen on dry ice, thawed at room temperature and resuspended in 10 \x\ of lysis buffer (20 mM Tris-HCI pH 7.5 containing 50 mM NaCI, 1 mM EDTA). The cells were disrupted with a Sonicator™ (Heat Systems-Ultrasonics, Plainview, NY) for 15 s on 30 setting 7, and cell debris was removed by centrifugation at 15,000 x

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g for 10 minutes. The protein concentration of each lysate was determined by measuring its absorbance at 280 nm. Proteins (100 ug) in each lysate were separated on a Tris-glycine 10-20% SDS-polyacrylamide gel, transferred to nitrocellulose and blocked
5 overnight in phosphate-buffered saline containing 0.05% Tween 20 (PBS-T) and 5% non-fat milk (w/v) at 4°C. An anti-GFP monoclonal antibody (Clontech, Palo Alto, CA) diluted 1:1000 in PBS-T containing 5% non-fat milk was used to probe the blot followed by incubation with a horseradish peroxides-coupled anti-mouse
10 secondary antibody (KPL, Gaithersburg, MD) diluted 1:2000 in PBS-T containing 5% non-fat milk. Protein-antibody complexes were detected using LumiGlo detection reagents (Cell Signaling Technology, Beverly, MA). The amount of GFP produced in E. coli was measured by densitometry using a molecular imager FX (Bio-
15 Rad, Hercules, CA) and Quantity One software.
Each PLAC4 variant was tested for its ability to drive E. coli expression of a reporter gene encoding GFP that was cloned in-frame with the S. cerevisiae a-mating factor pre-pro domain in
20 each of the pGBN vectors. The presence of GFP produced from PLAC4 variants in E. coli lysates was analyzed by Western analysis. Removal of the PBI sequence by mutation resulted in an 87% decrease in GFP expression (Figure 3A, lane 5), as determined by densitometry, relative to GFP produced by the wild-type PLAC4
25 (Figure 3A, lane 2). However, mutation of both PBII and PBIII
sequences (Figure 3A, lane 6) did not detectably down-regulate GFP expression. Deletion of all three Pribnow box-like sequences from PIAC4 by replacement with PPGKV DNA (Figure 3A, lanes 3 and 4) lead to a complete loss of detectable GFP expression. These results
30 indicate that the majority of PLAC4 expression in E. coli is dependent

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upon the presence of the PBI sequence.
Cloning and expression of enterokinase and HSA in K. lactis
5 Primers P9 and P10 were used to amplify the gene encoding
HSA that was subsequently cloned in frame with the a-MF sequence in the XhoI/NotI sites of the various pGBN vectors. Primer P9 was designed to encode the K. lactis Kex1 protease cleavage site immediately upstream of the HSA open reading frame to ensure
10 correct processing of the protein in the Golgi. K. lactis strains
containing integrated pGBN-HSA DNA were grown in 2 ml cultures of YPGal for 48 hours at 30°C. The level of HSA secretion was visually assessed by separation of 15 pi of spent culture medium on 10-20% Tris-Glycine gels followed by Coomassie staining. A DNA
15 fragment encoding the EKL was PCR amplified with primers Pll and P12 and cloned in-frame with the a-MF pre-pro domain in the Xhol/Bglll restriction sites of the various pGBN vectors containing the PLAC4 variants or in the vector pKLACl (see below). The DNA sequence of EKL in the various pGBN- EKL or pKLACl- EKL vectors
20 was confirmed by nucleotide sequencing. Secretion of enterokinase by K. lactis strains containing integrated pKLACl- EKL constructs was assessed by growing cells in 2 ml YPGal for 48 hours at 30°C and assaying spent culture medium for enterokinase activity as described below.
25
Enterokinase activity assay
Spent culture medium was isolated by microcentrifugation of 1 ml of a saturated culture of pKLACl- EKL integrated K. lactis at 30 15,800 x g for 1 minute to remove cells. Enterokinase activity was

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measured using the fluorogenic peptide substrate GDDDDK- 0-napthylamide (Bachem, King of Prussia, PA). Spent culture medium (50 pi) was mixed with 50 pi enterokinase assay buffer (124 mM Tris-HCI pH 8.0 containing 0.88 mM GD4K-(3-napthylamide, 17.6%
5 dimethylsulfoxide) and fluorescence intensity (excitation 337 nm, emission 420 nm) was measured over time. A comparison of the amount of enzyme activity associated with measured quantities of purified enterokinase (New England Biolabs, Beverly, MA) to the activity present in spent K. lactis culture medium was used to
10 estimate the amount of active enterokinase secreted by K. lactis strains. To compensate for a mild inhibitory effect that YPGal culture medium has on the enterokinase assay, purified enterokinase was first diluted into spent medium from a culture of untransfected K. lactis cells prior to measuring enterokinase activity as described
15 above.
PLAC4 variants retain full promoter activity in K. lactis
To test if the PLAC4 variants were able to direct expression of a 20 heterologous gene in K. lactis, the gene encoding HSA was cloned into each of the pGBINi vectors. HSA was chosen as a reporter protein due to its high expression and efficient secretion from K. lactis when expressed from wild-type PLAC4 (Fleer, et al. Bio. Technol. 9:968-975 (1991)). K. lactis strains containing each of the 25 integrated pGBNl-HSA expression vectors were grown to saturation in YPGal medium and secreted proteins in the spent culture medium were separated by SDS-PAGE and detected by Coomassie staining. HSA migrates as a 66 kDa band that can readily be detected in unconcentrated spent culture medium, and its identity was 30 confirmed by Western blotting with an anti-HSA antibody. K. lactis

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strains containing integrated pGBNlPB1-HSA, pGBNlHyb -HSA and pGBNlPBII-PBIII,-HSA vectors secreted HSA in amounts comparable to a control strain harboring pGBNl-HSA where HSA is expressed from wild-type pLAC4 (Figure 3B, lane 2). These data indicate that
5 mutation or deletion of the PBI, PBII and PBIII sequences of P^ does not significantly alter the promoter's ability to function in K. lactis. It is also noteworthy that markedly less HSA was secreted from cells harboring pGBNlPGKI-HSA (Figure 3B, lane 3) compared to cells expressing HSA from either wild-type PLAC4, (Figure 3B, lane 2)
10 or the other PLAC4 variants (Figure 3B, lanes 4-6). This is consistent with the notion that HSA expression from PPGKl is suppressed in galactose-containing medium because both UAS sequences required for galactose-induced expression have been deleted.
15 Effects of PLAC4, variants on the cloning efficiency of bovine enterokinase
Bovine enterokinase is a commercially important protease that is often used to cleave affinity tags from engineered fusion
20 proteins. Commercial production of enterokinase in E. coli is
plagued by low yields that are attributable to the protein's toxicity in bacteria.
Expression of enterokinase in K. lactis is shown here as a means to circumvent poor expression in bacteria. Numerous attempts to
25 assemble K. lactis expression vectors in E. coli, where DNA
encoding the EKL was placed downstream of wild-type PLac4 resulted in widespread isolation of clones containing loss-of-function mutations (e.g. frame shifts or early terminations) within the Encoding sequence. PLAC4 variants that exhibited reduced or abolished
30. expression in E. coli are shown here to facilitate cloning of the toxic

WO 2005/100586 PCT/US2005/011858
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EKL gene into K. lactis expression vectors in E. coli prior to their introduction into yeast. The EKL gene was PCR-amplified using a high-fidelity polymerase and cloned downstream of the various PLAC4 variants in the pGBNl vectors (see Figure 2B). The entire EKL gene
5 (708 bp) of numerous isolated clones was sequenced to determine the presence of loss-of-function mutations. When cloned under the control of wild-type PLAC4, in pGBNl, 11 of 12 (92%) clones examined contained loss-of-function mutations. However, no mutations were found in EKL cloned in vectors pGBNlPGKI (9 clones sequenced) or
10 pGBNlHyb (7 clones sequenced), vectors containing PLAC4 variants that completely lack E. coli promoter function. Additionally, no mutations were found in EKL cloned in vector pGBNlPBI (9 clones sequenced) where E. coli expression is reduced ~87% due to mutations in PBI. Additionally, 3 of 10 (30%) of EKL clones in pGBNlPBII.PBIII contained
15 loss-of-function mutations. Together, these data show that the function of wild-type PLAC4 in E. coli adversely affects the cloning efficiency of a toxic gene, and indicate that PLAC4 variants that either lack or have severely reduced function in E. coli are better suited for the assembly of K. lactis expression constructs in bacteria.
20
Construction of pKLAC1, an integrative K. lactis expression vector
A novel K. lactis integrative expression vector (pKLAC1) for
25 commercial secretion of proteins from K. lactis has been created. This vector is based on the PLAC4.pBI variant that contains mutations in PBI (see Figure 2B, pGBNlPB1) and contains (in 5' to 3' order): a PBI-deficient LAC4 promoter, the K. lactis a-mating factor secretion leader sequence, a multiple cloning site, the K. lactis LAC4 30 transcription terminator, a selectable marker cassette containing

WO 2005/100586 PCT/US2005/011858
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the Aspergillus nidulans amdS gene expressed from the PADHI AND an E. coli origin of replication and ampicillin resistance gene to allow for its propagation in E. coli.
5 Digestion of this vector with SacII or BstXI generates a linear
expression cassette that integrates into the promoter region of the PLAC4 locus of the K. lactis chromosome upon its introduction into K. lactis cells. Transformed yeast are isolated by nitrogen source selection on yeast carbon base medium containing 5 mM acetamide,
10 which can be converted to a simple nitrogen source only if the
expression cassette (containing the amdS gene) has integrated into the chromosome (U.S. Patent 6,051,431).
DNA encoding the K. lactis a-MF pre-pro domain was PCR-
15 amplified from K. lactis genomic DNA using primers 13 and 14 and cloned into the SacI/Xhol sites of pLitmus29 (New England Biolabs, Beverly, MA). The cloned K. lactis a-MF sequence was subsequently excised by Hindllll and Xhol digestion and cloned into the Hindlll/Xhol sites of plasmid pGBNlPBI to produce plasmid pGBNlPBI-
20 Kl α-MF. A 1520 bp DNA fragment containing all of the A. nidulans amdS gene except the first 128 bp was amplified using primers P15 and P16 and a cloned amdS gene as a template (DSM Biologics B.V., Delft, Netherlands). This fragment was cloned into the BamHI/Smal sites of plasmid pGBNlPB1-KI -MF replacing the G418
25 resistance gene and producing plasmid pGBNlPB1-KI a-MF-1520. The remaining 128 bp of the 5' end of amdS gene was amplified by PCR with primers P16 and P17, digested with BamHI, cloned into the BamHI site of vector pGBNlPBl-KI a-MF-1520 and the proper orientation of the fragment was confirmed by DNA sequencing. The
30 resulting vector is named pKLACl (GenBank Accession No.

WO 2005/100586 PCTAUS2005/011858
-22-
AY968582) and is commercially available from New England Biolabs, Beverly, MA.
Vector pKLAC1 was used to secrete enterokinase from K.
5 lactis cells after successfully assembling the expression vector in E. coli (PKLACI-EKL). Strains harboring integrated pKLAC1-EKL were cultured in YPGal medium for 2 days. Enterokinase proteolytic activity in the spent culture medium was assayed by measuring the rate of cleavage of a fluorogenic peptide. Measurements of activity
10 performed on culture supernatant from seven pKLACl-EKL integrated strains showed that all seven secreted active enterokinase (KLEK) (Figure 5). However, two of the seven strains (KLEK-S1 and KLEK-S4) secreted greater levels of enterokinase activity than the other five. Southern analysis determined that
15 strains KLEK-S1 and KLEK-S4 contained multiple tandem copies of integrated PKLACI-EKL. The yield of enterokinase secreted from strain KLEK-S1 grown in shake flasks was estimated to be ~1.1 mg/L based on a comparison of secreted enzyme activity to the activity of known quantities of purified enterokinase as described
20 above.

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TABLE 1. Oligonucleotides used in this study
Primer Sequence*
P1 5'-CTGTTACTCTCTCTCTTTCAAACAG-3'(SEQ ID NO:5)
P2 5,-GCATGTATACATCAGTATCTC-3 (SEQIDNO:6)
P3 5'-GGTATTrAATAGCTCGAATCAATGTGAGAACAGAGAGAAGATGTTCTTCCCTAACTC-3'
(SEQ ID NO:7)
P4 5'-GTMTGTTTTCATTGCTGTTTTACTTGAGATTTCGATTGAGAAAAAGGTATTTAATAGCTC
P5 GAATCAATG-3" (SEQIDNO:8)
P6 5V3TTTCTTAGGAGMTGAGAGCTCTmGTTATGTTGC-3' (SEQ ID NO:9)
P7 S'-GCAACATAACAAAAGAGCTCTCATTCTCCTAAGAAAC-S' (SEQIDNO:10)
P8 S'-GGAAGAEQIATGAGTAAAGGAGAAGAACTT-S' (SEQ ID NO:11)
P9 5'-ATAAGAATGCGGCCGCTTATTTGTAGAGCTCATCCATGCC-3' (SEQ ID NO:12)
P10 5'-CCGCTCGAGAAAAGAGATGCACACAAGAGTGAGGTTGCT-3' (SEQ ID NO:13)
P11 S'-ATAAGAATGCGGCCGCTTATAAGCCTAAGGCAGC-S' (SEQ ID NO:14)
P12 5'-CCGCTCGAGAAAAGAATTGTTGGTGGTTCTGATTCTAGA-3' (SEQ ID NO:15)
P13 5'-GGAAG£ICICTAATGTAGAAAACTTTGTATCC-3, (SEQIDNO:16)
P14 S'-TCCGAGCTCAAGCTTGAAAAAAATGAAATTCTCTACTATATTAGCC-S' (SEQ ID NO:17)
P15 5'-CCGCTCGAGATCATCCTTGTCAGCGAAAGC-3' (SEQIDNO:18)


WO 2005/100586

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24
WE CLAIM:
1. A method of producing a recombinant protein in yeast cells,
comprising: (a) obtaining a vector into which a gene encoding the target
5 protein has been inserted, the vector further comprising a modifiedPLAC4 wherein the modification results in a significant reduction in gene expression when the vector is cloned in bacteria; (b) transforming yeast cells with the vector; and (c) producing an effective amount of the recombinant protein in the yeast cells.
10
2. A method according to claim 1, wherein the yeast cells areK. lactis yeast cells.
3. A method according to claim 1, wherein the bacteria are E. coli.
15
4. A method according to claim 1, wherein the modifiedPLAC4 has a
mutation in one or more Pribnow box-like sequences.
5. A method according to claim 4, wherein the one or more Pribnow
20 box-like sequences are PBI, PBII and PBIII.
6. A method according to claim 5, wherein the mutation is in two or
more Pribnow box-like sequences.
25 7. A method according to claim 1, wherein the modified PLAC4 has one or more mutations in a first region of the promoter corresponding to nucleotides -198 to-212.
8. A method according to claim 1, wherein the modifiedPLAC4 has one 30 or more mutations in a second region of the promoter corresponding to nucleotides-133 to-146.

WO 2005/100586 PCTYUS2005/011858
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9. A method according to claim 1, wherein the modifiedPLAC4 has one
or more mutations in the first region and the second region of the
promoter.
5
10. A method according to claim l, wherein nucleotides-1 to-283 in the
modifiedPLAC4 are substituted by nucleotides-1 to-283 of a
phosphoglycerate kinase promoter from S. cerevisiae.
10 11. A method according to claim 1, wherein the vector in the transformed yeast cells is an episomal plasmid.
12. A method according to claim 1, wherein the vector in the
transformed yeast cells is an integrative plasmid.
15
13. A method according to claim 1, wherein the vector contains a DNA
sequence encoding a yeast secretion signal peptide.
14. A method according to claim 1, wherein the vector contains a DNA
20 sequence encoding a selectable marker.
15. A method according to claim 1, wherein at least 50% of the
transformed yeast cells express recombinant protein.
25 16. A DNA vector comprising: a modified PLAC4 promoter.
17. A DNA vector according to claim 16, further comprising aK. lactis a-mating factor.

WO 2005/100586

26

PCT/US2005/011858

18. A DNA vector according to claim 16, further comprising an A. nidulans
acetamidase selectable marker gene.
19. A DNA vector according to claim 16, further comprising a multiple
5 cloning site for insertion of a gene encoding a recombinant protein.
20. A DNA vector according to claim 16, further comprising a PLAC4
terminator.
10 21. A host yeast cell, comprising the vector of claim 16.
22. A host bacterial cell, comprising the vector of claim 16.
23. A kit, comprising: a vector according to claim 16; optionally 15 competent yeast cells; and instructions for use.
24. A modified PLAC4 Pribnow box wherein TTATCATTGT (SEQ ID NO: 22)
is modified to AGAACAGAGA (SEQ ID NO: 23) and TATTATTCT is modified
to GAGAGCTCT.
20
25. A method of producing recombinant protein in yeast cells, a DNA
vector, a host yeast cell, a host bacterial cell, a kit and a modified PLAC4
Pribnow box, substantially as herein described with reference to the
accompanying examples and figures.
25
Dated this 11th day of October, 2006.

[RAJESHWARI H.]
OF K&S PARTNERS
ATTORNEY FOR THE APPLICANT

WO 2005/100586

PCT/US2005/011858

ABSTRACT
Title: "Method for construction and use of Kluyveromyces Lactis 5 Promoter Variants in K. Lactis that substantially lack E. Coli Transcriptional capability"
Methods and compositions are provided relating to production of 10 recombinant protein in yeast. A modified PLAC4 is described where one or more mutations may be introduced into the Pribnow box-like sequences in the promoter. The modified promoter when placed upstream of a target gene in a vector causes a significant reduction of target gene expression in transformed bacteria but produces efficient 15 expression of the target gene in yeast.

WO 2005/100586

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NEB-246-PCT.ST25.txt
SEQUENCE LISTING
New England Biolabs, Inc. Taron, Christopher Colussi, Paul
Method for construction and use of Kluyveromyces lactis Promoter Variants in K. lactis that substantially Lack E. coli Transcriptional Capability
NEB-246-PCT
60/560,418 2004-04-08
23
Patentln version 3.2
1
24
DNA
unknown

relevant segment of the LAC4 promoter of Kluyveromyces lactis
1
caatgtgtta tcattgtgaa gatg 24
2
26
DNA
unknown

relevant section of LAC4 promoter of Kluyveroyces lactis
2
gagaattatt attcttttgt tatgtt 26
3
24
DNA
unknown

Sequence reflecting mutated bases in LAC4 promoter of K. lactis in vector

mutation
(8).. (9)

mutation
(11)..(11)

mutation
Page 1

WO 2005/100586

PCT/US2005/011858

NEB-246-PCT.ST25.txt (14)..(15)

mutation
(17)..(17)
3
caatgtgaga acagagagaa gatg 24
4
26
DNA
unknown

sequence reflecting mutated bases in LAC4 promoter of K. lactis in vector

mutation
(7)..(7)

mutation
(9)..(12)
4
gagaatgaga gctcttttgt tatgtt 26
5
25
DNA
unknown

primer
5
ctgttactct ctctctttca aacag 25
6
21
DNA
unknown

primer
6
gcatgtatac atcagtatct c 21
7
55
DNA
unknown

primer
Page 2

WO 2005/100586

PCT7US2005/011858

NEB-246-PCT.ST25.txt
7
ggtatttaat agctcgaatc aatgtgagaa cagagaagat gttcttccct aactc 55
8
70
DNA
unknown

primer
8
gtaatgtttt cattgctgtt ttacttgaga tttcgattga gaaaaaggta tttaatagct 60
cgaatcaatg 70
9
37
DNA
unknown

primer
9
gtttcttagg agaatgagag ctcttttgtt atgttgc 37
10
37
DNA
unknown

primer
10
gcaacataac aaaagagctc tcattctcct aagaaac
11
30
DNA
unknown

primer
11
ggaagatcta tgagtaaagg agaagaactt
12
40
DNA
unknown

primer
Page 3

WO 2005/100586
NEB-246-PCT.ST25.txt 12 ataagaatgc ggccgcttat ttgtagagct catccatgcc
13
39
DNA
unknown

primer
13
ccgctcgaga aaagagatgc acacaagagt gaggttgct
14
34
DNA
unknown

primer
14
ataagaatgc ggccgcttat aagcctaagg cage
15
39
DNA
unknown

primer
15
ccgctcgaga aaagaattgt tggtggttct gattctaga
16
32
DNA
unknown

primer
16
ggaagatctc taatgtagaa aactttgtat cc
17
46
DNA
unknown

primer
17
tccgagctca agcttgaaaa aaatgaaatt ctctactata ttagcc
Page 4

WO 2005/100586

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NEB-246-PCT.ST25.txt
18
30
DNA
unknown

primer
18
ccgctcgaga tcatccttgt cagcgaaagc 30
19
77
DNA
unknown

primer
19
cggggatcct ttcagaggcc gaactgaaga tcacagaggc ttccgctgcg gatcttgtgt 60
ccaagctggc ggccgga 77
20
36
DNA
unknown

primer
20
tcccccgggc tatggagtca ccacatttcc cagcaa 36
21
36
DNA
unknown

primer
21
cgcggatccg ccaccatgcc tcaatcctgg gaagaa 36
22
10
DNA
unknown

relevant section of LAC4 promoter of K. lactis
22
ttatcattgt 10
Page 5

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NEB-246-PCT.ST25.txt 23 10 DNA unknown

modified section of LAC4 promoter of k. lactis
23
agaacagaga 10
Page 6

Documents:

1219-MUMNP-2006-ABSTRACT(12-10-2006).pdf

1219-MUMNP-2006-ABSTRACT(28-12-2010).pdf

1219-MUMNP-2006-ABSTRACT(AMENDED)-(28-12-2010).pdf

1219-MUMNP-2006-ABSTRACT(GRANTED)-(30-3-2011).pdf

1219-mumnp-2006-abstract.doc

1219-mumnp-2006-abstract.pdf

1219-MUMNP-2006-CANCELLED PAGES(22-3-2011).pdf

1219-mumnp-2006-claims(amended)-(18-1-2011).pdf

1219-MUMNP-2006-CLAIMS(AMENDED)-(19-1-2011).pdf

1219-MUMNP-2006-CLAIMS(AMENDED)-(22-3-2011).pdf

1219-MUMNP-2006-CLAIMS(AMENDED)-(28-12-2010).pdf

1219-MUMNP-2006-CLAIMS(GRANTED)-(30-3-2011).pdf

1219-MUMNP-2006-CLAIMS(MARKED COPY)-(22-3-2011).pdf

1219-mumnp-2006-claims.doc

1219-mumnp-2006-claims.pdf

1219-mumnp-2006-correspondence(12-3-2008).pdf

1219-MUMNP-2006-CORRESPONDENCE(18-1-2011).pdf

1219-MUMNP-2006-CORRESPONDENCE(19-05-2010).pdf

1219-MUMNP-2006-CORRESPONDENCE(2-4-2012).pdf

1219-MUMNP-2006-CORRESPONDENCE(22-3-2011).pdf

1219-mumnp-2006-correspondence(8-1-2007).pdf

1219-MUMNP-2006-CORRESPONDENCE(8-7-2010).pdf

1219-mumnp-2006-correspondence(ipo)-(19-1-2010).pdf

1219-MUMNP-2006-CORRESPONDENCE(IPO)-(30-3-2011).pdf

1219-mumnp-2006-correspondence-received.pdf

1219-mumnp-2006-description (complete).pdf

1219-MUMNP-2006-DESCRIPTION(GRANTED)-(30-3-2011).pdf

1219-MUMNP-2006-DRAWING(12-10-2006).pdf

1219-MUMNP-2006-DRAWING(28-12-2010).pdf

1219-MUMNP-2006-DRAWING(GRANTED)-(30-3-2011).pdf

1219-mumnp-2006-drawings.pdf

1219-mumnp-2006-form 1(8-1-2007).pdf

1219-MUMNP-2006-FORM 18(13-3-2008).pdf

1219-MUMNP-2006-FORM 2(GRANTED)-(30-3-2011).pdf

1219-MUMNP-2006-FORM 2(TITLE PAGE)-(12-10-2006).pdf

1219-MUMNP-2006-FORM 2(TITLE PAGE)-(GRANTED)-(30-3-2011).pdf

1219-MUMNP-2006-FORM 26(28-12-2010).pdf

1219-MUMNP-2006-FORM 3(19-05-2010).pdf

1219-mumnp-2006-form 3(8-1-2007).pdf

1219-mumnp-2006-form-1.pdf

1219-mumnp-2006-form-2.doc

1219-mumnp-2006-form-2.pdf

1219-mumnp-2006-form-3.pdf

1219-mumnp-2006-form-5.pdf

1219-mumnp-2006-form-pct-isa-220.pdf

1219-mumnp-2006-form-pct-isa-237.pdf

1219-MUMNP-2006-OTHER DOCUMENT(8-7-2010).pdf

1219-mumnp-2006-pct-request.pdf

1219-mumnp-2006-pct-search report.pdf

1219-MUMNP-2006-REPLY TO EXAMINATION REPORT(19-1-2011).pdf

1219-MUMNP-2006-REPLY TO EXAMINATION REPORT(28-12-2010).pdf

1219-MUMNP-2006-SEQUENCE LISTING(28-12-2010).pdf

1219-MUMNP-2006-SEQUENCE LISTING(30-3-2011).pdf

1219-mumnp-2006-wo international publication report a2(12-3-2008).pdf

abstract1.jpg


Patent Number 247182
Indian Patent Application Number 1219/MUMNP/2006
PG Journal Number 13/2011
Publication Date 01-Apr-2011
Grant Date 30-Mar-2011
Date of Filing 12-Oct-2006
Name of Patentee NEW ENGLAND BIOLABS, INC.
Applicant Address 240 County Road, Ipswich, MA 01938,
Inventors:
# Inventor's Name Inventor's Address
1 Christopher Taron 36 Belcher Street, Essex, MA 01929,
2 Paul Colussi 873 Washington Street, Gloucester, MA 01930,
PCT International Classification Number C12P21/06 C12N1/18
PCT International Application Number PCT/US2005/011858
PCT International Filing date 2005-04-08
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/560,418 2004-04-08 U.S.A.