Title of Invention	A MEHTOD FOR ESTRACTING A RECOMBINANT NON-MEMBERANOUS PROTEIN INCLUDING PROINSULIN
Abstract	ABSTRACT This invention relates to a method of extracting a recombinant protein from a recombinant gram negative bacteria without tysing. It consists the steps of permeabilizing the cell membrane with a detergent, solubilizing the recombinant protein and the cell membrane with a denaturant and physically separating the recombinant protein from the cell membrane.

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This invention relates to a method for extracting a recombinant protein from within a recombinant gram negative bacteria. Several publications are referenced in this application. Full citation to these publications is found at the end of the specification, immediately preceding the claims, or where the publication is mentioned; and each of these publications is hereby incorporated by reference. These PATENT 540519-2003 publications relate to the state of the art to which the i nventi on pertains; however, there is no admission that any of t" h^so pub! i cat ions is indeed prior art . BACKGROUND OF THE INVENTION Recombinant DNA technology has enabled the expression of foreign (heterologous) proteins in microbial and other host '(■I 1 R . A vector containing genetic material directing the host r I 1 to produce a protein encoded by a portion of the ]i"t frol orjous PNA sequence is introduced into the host, and the i r a (informant host cells can be fermented and subjected to cm idii i• u in whi ch facilitate the expression of the heterologous PNA, 1 C'lil i ng to the formation of large quantities of the desired pi" i The advantages of using a recombinantly produced pi'itoin in lieu of isolat ion from a natural source include: the ready availability of raw material; high expression levels, which i n especially useful for.proteins of low natural abundance; the o.ir.G with which a normally intracellular protein can be excreted i nl o th'1 express i on medium, facilitating the purification process; and the relative ease with which modified (fusion) prote i nr; can be created to further simplify the purification of t fjo rTiul Cant protein. However, the aforementioned benefits of recombinant DNA technology are also accompanied by several disadvantages,, namely: the required elements of the active protein which result from PATENT 540519-2003 poitt t -rnnrsl ational modification (i.e., glycosylation) may not be '•■iiii C'i\ out in the expression medium; proteolytic degradation of iK-wly formed protein may result upon expression in host cells; ."it r] i hr> formation of high molecular weight aggregates, often rTved to an "inclunion bodies" or "refractile bodies", which r tvsiil t from the inabil ity of the expressed proteins to fold forrorMy in an unnatural cellular environment. The recombinant prnin i n r.innot be excreted into the culture media upon formation of i ncl ufi i on bodies. Indus i on bodies contain protein in a stable non-native fonf nrmai" ion; or, the protein aggregates may be amorphous, comprised of partially and completely denatured proteins, in i n."i ecu rate translation. Such inclusion bodies constitute a large p^rt ion of the total cell protein. Inclusion bodies present significant problems during the purification of recombinant proteins, as they are relatively insoluble in aqueous buffers. Denaturants and detergents, i.e., gunnidinn hydrochloride, urea, sodium dodecylsulfate {SDS}'and Tri ton X -100, may be necessary to isolate the proteins from the inclusion bodies, often at the expense of the biological activity of the protein itself, resulting from incorrect folding and modification of the amino acid residues in the sequence. Additionally, a result of the expression of recombinant onA in E. coli is the accumulation of high concentrations of ■PATENT ■ .540510 2003 acetato in the media, mainly during the induction phase. The deleterious effect of acetate accumulation (greater than 5g/L) on cell growth and recombinant protein expression has been well documented in the literature. Further, the recovery of the desired protein from inclusion bodies is often complicated by the need to separate the d".ni red protein from other host cellular materials, in addition to rsopgr.it ing the desired protein from inclusion body hotorologous protein contaminants. The latter problem results f mm the filrong attraction that inclusion body proteins have for -mo ,-mot.hor, due to strong ionic and hydrophobic interactions. i Consequently, most established protocols for the i col nt. i on of recombinant proteins from inclusion bodies result in l.itq^ quantities of biologically inactive material, and very low y i • ■ 1 i\r, of net ive protein, uncontaminated by extraneous ][,,(■ oyoloqous protein. Researchers have focused on the manipulation of phage i n order to stimulate protein synthesis by a variety of methods. The promoters of the Lambda phage (PL and Pv) are i E-.\ rong promoters that are negatively controlled by the repressor rorl'-'d by the gene cl. The mutation cl857 renderedf the repressor imctivnte at temperatures above 37'C. Thus, the expression of a sequence controlled by these' promoters and by the repressor cl957 can be activated by a simple change in temperature. These promoters are often used in E. coli expression vectors, because they are strong and efficiently repressed (Denhardt & Colasanti, 1987) . ; Remaut et al. (1981) constructed a set of plasmicis containing the promoter PL. The promoter and the trp region of the gene were taken from a family of phages (trp44) and inserted in the plasmid pBR322, creating the first plasmid of! a series, ! ' I !' . . plasmid pPLa2. After several manipulations, other plasmida were I obtained. The plasmids pPI.a2 and pPLaS contained the promoter PL i fragment, the origin of replication, the ampicillin resistance i i qori" from the plasmid pBR322, and a kanamycin resistance gene fmm the plasmid pMK20. The promoter region contained the promoter/operator and the nutL site (antitermination), but it was Inching the beginning of the gene N. The plasmids pPLc236, pPLc28 and pPLc24 are different ft OTM the previously identified plasmids, with respect to the direction of transcription from the promoter PL in relation to thn orientation of the origin of replication, as found in pBR322 (n - anticlockwise, c = clockwise). The kanamycin resistance gone is absent in these three vectors. The difference between pPLc236 and pPLcS is the presence or absence of a region (present in the former and absent in the latter), which affects the region of uniquo cloning sites. pPLc24 was derived from pPLc28 by insertion of a region containing the ribosome binding site of the genp for replicase from the phage MS2, enabling the expression of eukaryot ic genes. These plasmids were tested with the expression of different genes, e.g., the gene trpA from Salmonella typhimurium, cloned in the plasmid pPTic23 {predecessor of pPLc236) , which * nhowed 40% induction of product in relation to the total cellular protein. pL.c236 programmed, in E. coli resulted in a expression of the gene ROP as 20% of the total protein {Muesing et al., 1984}. The proteins p4 and p3 of the phage 29 of Bacillus niibtilis, were also produced from pPLci and reached 30% and 6%, respectively, of the total cellular protein induced in E. coli, after thermal induction (Mellado & Salas, 1982). In 1983, Remault et al. (1983a) built a plasmid i pPLc245, derived from pPLc24, in which the initial coding region of replicase was deleted and a region with several unique cloning-sites was added, permitting direct expression. The gene i for human a-interferon was cloned into this plasmid, resulting in induction of protein of approximately 2% to 4% of the total rrMlular protein. For c*-interferon, the levels of expression varied from 3% to 25% of the cellular protein, depending on the plasmids used, e.g., pPLc245, pPLc28 and pCP40, and on:the pi-oponce of a transcription-terminator from phage T4 (Simon's et ■i 1 . , 1381). The plasmid pCP40, derived from pPl«, ■ was , built by K^maut et al. (1983b). The promoter-region was transferred to a plnRmiri derived from pKN402 with temperature dependent 'runaway' replication. When the cultures are heated to 42DC, the repressor c]'"' in deactivated and the promoter PL is liberated, resulting in an increase in the number of copies of the plasmid pCJMO, by approximately ten fold. Crowl et al. (1985) relates to four plasmids containing the promoter PL. The plasmid pRC23 was built containing the promoter PL and a synthetic Shine-Dalgarno region, without the codon ATG, cloned in the pla,smid pRC2 (derived from pBR322) . To build the other three plasmids, pEV-vrfl, pEV-vrf2 and pEV-vrf3, a region with unique cloning sites was inserted, adding the initial ATG codon, such that in each one the reading fram.es are on phase. The plasmid pRC23 was used for the expression of intorleucine-2 and cv-interf eron, with a level of 10% to 20% of the total cellular protein. Lautenberg et al. (1983) built the plasmid pJL6, containing the promoter Plp, which codes for initiation of translation of the gene ell of the phage, with uniijue Clal and /h'ndlTI cloning sites, located at 50 bp from the initial ATG site. Cones, adequately cloned in these sites are; induced, 1 i' producing fusion proteins with the protein CII. Seth et al. (1986) modified this vector so that the induction of proteins I could occur without fusion.;. Three plasmids were'constructed, containing a Kpnl site in pANK-12, an Hpal site in'pANH-1, and an i NdnT s i to in pPL2 of the initial codon ATG of the gene. CIl of pJL6. In pANH-1, the amino acid 'valine' occurred more frequently in the amino-end of the induced protein. Production of oncogenes was obtained from these vectors. Chang and Peterson (1992) also modified the plasmid pJL6 and built a line of plasmids, pXC, in which the region for initiation of translation of the gene CII was substituted by a synthetic one. Additionally, a region was inserted having several unique cloning sites. The region CII affects the efficiency of the translation if the expression is required wi L hout fusion. With the synthetic region, the efficiency rose between 10 and 20 times, depending oh the spacing region between PD and ATG. The expression reached 48% of the total cellular protein for the protein 14 -31- 3 of cow brain, of which the DNA had been amplified by PCR. Schauder et al. (1987) built a line of plasmids derived from pJLS, containing the promoters P„ and PL in tandem, the region SD of the gene atpE (for subunit of ATPase), with the transcription terminator of the bacteriophage fd and with the gene of the repressor clB". These plasmids were named pJLA501 to [ i ■ -05 and differ in the regions of the multiple cloning sites. On testing the expression of the gene atpA (for a subunit of : ATPase), an induction of 50% of the total cellular protein was i1 found. The genes sucC and sucD, respectively, showed 30% and 15% induced protein in relation to the total cellular protein. Rosenberg et al. (1983) built the plasmid pKC30 and its derivatives. The vector pKC30 is used for the expression of i h barterial genes containing their proper translation-regulation regi our,. This vector contains a unique cloning Hpal site, located 321 bp downstream from the promoter PL, within the coding ipg ion o£ the gene N. The expression of the activator CII and ei ght mutants in just one amino acid was achieved in the vector pKC30. Because CII is quickly recycled in E. coli and deleterious for cell growth, with insertion and expression of its gone in pKC30, levels of 3% to 5% of the total cellular protein were reached. The production of the protein CII rose when the protein N (anti-terminator) was provided by the host-cell, because of the presence of the 'upstream' sequences of the gene CII of the sites nuth, nutR (for anti-termination) and trl (for termination). Other proteins were expressed from pKC30, such as the protein B of the phage Mu (Chaconas et al., 1985) and the protein UvrA of E. coli expressed at levels of 15% and 7% of the. total cellular protein respectively. For the expression of eukaryotic genes the plasmid i I i . pASl, derived from pKC30, was built with the cloned gene "CII. The complete coding region of CII was deleted and a' SamHI site wan added immediately 'downstream' of the ATG initiation codon. In tliis manner, the regulating regions for translation were rm intainod in the vector and a eukaryotic or synthetic gene can bo expressed if cloned correctly to the BamHI site. Expression of the gene for the antigen t of the virus SV40 resulted in this vwtor in levels of 10% of the total cellular proteins, after one hour of thermal induction (Rosenberg et al., 1983). Lowman et al. (1988) modified the plasmid pASl, .in iuunLing a Ncol site in the initial ATG, creating the plasmid ■ named pASl-N. Expression of the gene CAT and fusion with proteins of the virus SV40 were obtained. Later, Lowman & Bina (1990) uned these products to study the effect of temperature in L hermal induction. Mott et al. (19Q5) used pKC30 and pASl to express the hnoterial gene rho and verified that the thermal induction did n-~4" result in high levels of expression of the protein Rho • Induction with nalidixic acid and mitomicina C was tested in the hont-, f'T, which provoked the induction of the syntheses of Rec a, resulting in an inactivation of the repressor cl. In this mruiner, levels of expression varying from 5% to 40% of the cellular protein were reached. Hence, the manipulation of plasmids for expression of a prot-.ein or peptide of interest is a developing area and a method r for the induction of complex proteins such as pro-insulin via manipulation of a plasmid and a plasmid therefrom, have not; hejntofore been developed or suggested. ; U.S. Patent No. 4,734,362, to Hung et al.', is directed to a method of isolating polypeptides produced recombinantly in inclusion bodies. The disclosed method includes the cell lysis, and recovery of inclusion bodies comprising the desired mroinbinant protein, solubilization with denaturant, protection of the nulfhydryl groups of the recombinant protein, derivat ization of cationic amino groups of the protein, ana recovery of the derivatized recombinant protein. Olson, U.S. Patent No. 4,518,526 relates to a method of releasing active proteins from inclusion bodies by cell lysis, rentri f ugat ion, denaturation and renaturation. The patent tea chess the necessity of the disruption of the cell to separate the noluble and insoluble protein, followed by treatment of the insoluble fraction with a strong denaturant, and recovery of the renntured heterologous protein. ( ! Rausch, U.S. Patent No. 4,766,224 is directed to a method of purification and solubilization of proteins produced in transformed microorganisms as inclusion bodies. The purification is effected by solubilization of the inclusion bodies in detergent, treatment with a strong denaturant, followed by chromatographic separation to obtain renatured active protein. Builder et al., U.S. Patent No. 4,620,948:is concerned i ! with a process for isolating and purifying inclusion bodies by lysing the cell culture, precipitation of protein, denaturation of the insoluble fraction, and renaturation to ^solate the ; r retractile protein. ■ Similarly, U.S. Patent Nos. 4,734,368, 4,659,568, 4,9(12,783, 5,215,896, and EP 337,243 and WO 87/02673 are each directed to methods of purifying proteins entrapped in inclusion |v).jjnn, These methods use of the following techniques (alone or in eombination}: cell lysis,' denaturation, chromatographic npparnt ion, centrifugation, manipulation of the rkMiat-urat i nn/renaturation of the protein, and the attachment of 3 p,idor pept idea which facilitate the separation of the proteins from the i inclusion bodies . Rach of the aforementioned prior art processes utilize mrihods which disrupt the cell to release the inclusion bodies from t he? cellular material. There is no teaching or suggestion of a means for isolating inclusion bodies from cellular material without the disruption of the cell, nor is there a motivation to derive such a method from the teachings of the prior art. However, the lysis or disruption of cells is disadvantageous as it allows contaminants to be present with the desired protein, sur-h as lipopolysaccharides, which are very difficult to.separate from the desired protein. U.S. Patent Nos. 4,877,830, 5,115,102, 5,310,663, and RP f^6,419, WO 91/11454, WO 91/16912, WO 94/07912, Proc. Natl. I Acid. Sci. (1991) 88 (20), and Mol. Biol. Rep. (1993) 18_: 223-230 i i i are each directed to affinity purification of proteins. |These r documents relate to the use of (alone or in combination): metal chelate affinity chromatography for chromatographic separation of ' i i proteins having neighboring histidine residues, immunoaffinity r~h romatography, and the use of amino acid mimetics as eluents in affinity purification of proteins. , U.S. Patent Nos. 4,766,2 05, 4,599,197, 4,923,967, and RP ^12,lr^, EP 302,469, Biochemistry (1968), 7. (12), 4247, and J. ■Biological Chemistry (1959), 234 (7), 1733 are each directed to methods of sulfitolysis, i.e., the treatment of a protein, aolubi1ized in a strongly denaturing solution, with a mild oxidant in the presence of sulfite ion, which converts cysteine and cystine residues to protein-S-sulfonates. The strongly denaturing solution is weakened to permit refolding,'and ■ disulfide linkages are reformed using a sulfhydryl compound, in the presence of the corresponding disulfide (oxidized) form. Similarly, EP 208,539 and WO 87/02985 are directed to methods of facilitating protein refolding in vitro. EP 264,250, GB 2,067574, EP 055,945, MMW (1983) 1£5. (5?.), 14, J. Biol. Chem. (1971) 24_£ (22), 6786-91, J. Chrom. (1989) 461: 45-61 are each directed to insulin, its production frnm pro-insulin, and the purification of insulin and pro-insulin. U.S. Patent No. 4,578,355, to Rosenberg, is directed to the derivation and use of the PL transcription unit. EP 363,896 is directed to the use of ultrafiltration in protein purification. Human insulin, a proteolytic digestion product of pro-insulin, is a polypeptide hormone produced by beta cells of the islets of Langerhans in the pancreas. Its purpose is to decrease the amount of glucose in the blood by promoting glucose uptake by cellr, and increasing the capacity of the liver to synthesize glycogen. The action of insulin is antagonistic to glucagon, ^t£5±SH2EIl adrenal glucocorticoids and adrenaline, and its deficiency or reduced activity produces diabetes with a raised blood sugar 1evel. Human insulin has been prepared from several sources, including: isolation from human pancreas, peptide synthesis, the semi fsynfchet ic conversion from porcine insulin and fermentation of F.. col i bacteria or Saccharomyces cerevisiae yeast, suitably oncodod by DNA recombinant methods. These methods suffer from poor yield and cost efficiency, and the development of a high yielding, cost effective method of producing human insulin for the treatment of diabetes has been the subject of much research efforts in recent years. Hence, a method for the induction of human pro-insulin via recombinant techniques has not heretofore been! realized, i wherein the protein may be isolated in substantial1 quantities from inclusion bodies, especially such a method wherein cell lysis or cell disruption is avoided. OBJECTS AND SUMMARY OF THE INVENTION Objects of the present invention may include providing at least one of: a vector comprising at least one [nucleic acid such as DNA for cloning of a nucleic acid or f ;r expression of at lpast one heterologous protein by a cell such as Gram negative bacteria (the vector can comprise a nucleic acid molecule, e.g., i DMA, encoding: an origin of replication region, optionally, and preferably a selection marker (which can be a coding nucleic acid Pftl'KN'i- in a restriction site), a promoter, an initiation region e.g. a translation initiation region and/or a ribosome binding site, at leant one restriction site for insertion of heterologous nucleic ncid, e.g., DNA, encoding the heterologous protein, and a transcription terminator); :-a method for extracting a recombinant protein from within a cell such as a recombinant Gram negative I'aripria having a cell membrane; and, a method for purifying an i r>ol nr pd recombinant human insulin . Accordingly, the present invention provides a vector (-■nmprisinq at least one nucleic acid molecule such as DNA for cloning of a nucleic acid molecule, or more preferably, for ^xpremsion of at least one heterologous protein by cell such as a nr.im negat ive bacteria. The vector can comprise DNA encoding the following: an origin of replication region, optionally and profp-rably a selection marker (which can be coding DNA in a restriction site), a promoter, an initiation region e.g. a trr.Tnsl.it ion initiation region and/or a ribosome binding site) at least one restriction site for insertion of heterologous DNA encoding the heterologous protein, and a transcription terminator. The Gram negative bacteria can be E. coli. The origin of replication region can be from plasmid pUC8. The initiation region can be a translation initiation region and can be synthetic, e.g., synthetic Shine-Dalgarno regions from gene 10 of plinne T7. The selection marker can be a tetracycline resistance P&TERT mnrker. Alternatively or additionally, selection of transformed cr>lis containing the vector can be on the basis of a product expressed by the heterologous DNA encoding the heterologous proioin. The promoter can be a PL promoter. And, the transcription terminator can be a Rho-independent one. Thus, the invention can provide a vector comprising DNA for expression of a heterologous protein by a Gram negative bacteria. The vector can comprise at least one nucleic acid molecule, e.g., DNA, encoding the following: an origin of replication region, a selection marker, a promoter,- a translation i ni t. i at i on region or a ri bo some binding site, at least one i restriction site for insertion of heterologous DMA encoding the heterologous protein, and a transcription terminator. The DNA encoding the at least one restriction site preferably encodes multiple restriction sites; and, the multiple rontrict ion sites are preferably Ncol, £coRI, StuI, PstI,' SamHI, nnd BspF,l. | i The present invention further provides a vector for expression of a pro-insulin by a Gram negative bacteria. That ir,, in the inventive vector, at the at least one restriction site for insertion of heterologous DNA (or a heterologous nucleic acid sequence) there can be inserted a nucleic acid molecule such as DNA encoding pro-insulin, e.g., human pro-insulin. The protein expressed by the inserted nucleic acid molecule, e.g., pro-i nsulin such as human pro-insulin, can contain tag or a marker, for instance, a His tag (which is useful for separating, i no]at ing and/or purifying the protein) . And therefore, more generally, inventive vectors can include at least one exogenous coding nucleic acid molecule at the at least one restriction site for insertion of a heterologous nucleic acid molecule, e.g., exogenous coding DNA can be at the at least one restriction site for insertion of heterologous DNA. FurMier, the exogenous coding DNA can encode, in addition to the heterologous protein, a marker or tag, for instance a Hia tag. Still further, the invention provides a method for extract ing a recombinant protein such as pro-insulin, e.g., human pro-insulin, from within a cell such as a recombinant Gram neqq t i ve bacteria having a cell membrane, without lysing the bn (a) permeabilizing the cell membrane by contacting the ■ bacteria with a detergent under conditions which facilitate the extraction of native cell proteins from the cell membrane without extracting the recombinant protein from the i"p] 1 membrane ,- (b) solubilizing the recombinant protein and cell membrane; and (c) separating the recombinant protein from the cell membrane. Accordingly the present invention provides a method for extracting a recombinant non-membranous protein including pro-insulin from within inclusion bodies of a recombinant gram negative bacteria having a cell membrane, without lysing the bacteria comprising the steps of: (a)permeabilizing the cell membrane by contacting the bacteria with a detergent such as herein described to separate native cell proteins from the cell membrane without separating the recombinant protein from the cell membrane; (b)solubilizing the recombinant protein and the cell membrane in a known manner, and (c) separating the recombinant protein from the cell membrane in a known manner. BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES: In the following detailed description reference will be made to the accompanying drawings, incorporated herein by reference, wherein: Fig. 1 shows a construction of the plasmid pUTc6 containing the gene of tetracycline resistance of plasmid pRP4 and the origin of replication pUC8 with only one EcoRI cloning site. Fig. 2 shows a construction of plasmid pULTDK 7.1 containing the P,, promoter of phage lambda and the Shine-Dalgarno region of gene 10 of phage T7; Pig. 3 shows a polylinker addition in pUTC6 and subsequent cloning of the fragment containing the PI promoter and Shine-Dalgarno region of plasmid pULTDK 7.1 and a construction of pT.HC 8.1; Pig. 4 shows a final construction of hyperexpression vector pIiMT8.5 by addition of the synthetic transcription terminator in pI,MC8 .1; Pigs. 5 and 5A shows a map of vector pLMT8.5; i i Pig. 6 shows the results of direct inclus4°n body i no! nhil i y.nt ion of pre-treated cells with 8M urea versus time i (Innnn 1-2 represent 6 hours of solubilization; -lanes 3-4 represent 8 hours of solubilization; and lanes 5-6 represent 24 hours of solubilization, wherein M denotes molecular weight i i : marker, S denotes supernatant and P denotes residual pellet); Fig. 7 shows the purification of pro-insulin fusion prnt-piri by pFI precipitation, wherein aliquots of solubilized {8M i h . ■ urr-n) and dialyzed fusion protein were precipitated'at different pll values (at pH 4.5, all the recombinant protein could be recovered in the precipitate (lane 5); lanes 1 to 7 represent pH values of 5.5, 5.5, 5.0, 5.0, 4.5, 4.5, 4.0 and molecular- weight m.ukor, respectively, with P referring to pellet and S referring ln nnpprnarant); Fig. a shows a schematic representation of the invntivp process for isolating recombinant human insulin; Fig. 9 shows an analysis in a 15% denaturing gel of t-nt-nl collular protein from cultures transformed with pLMT8.5, pPTAl or pPLT4.1, at different induction times at 40oC {the arrow indicates the induced recombinant pro-insulin protein); Figs. 10A-M show the nucleotide sequence of pLMT8.5 and thn positions of restriction, endonuclease sites; Figs. 11A-F show a tabulation of the restriction sites in the sequence of pLMT8.5 and the length of the restriction fragment produced; Figs. 12 and 13 show the strategy for obtaining a fragment from pLA7 containing the pro-insulin sequence and a i liiotidine tag and inserting them into the restriction site of the multiple cloning sites of vector plasmid pLMT8.5 to yield vector plnsmid pHIS, and ; Figs. 14 and 15 show construction of pPLT4 and sequence containing lead of T7. DETAILED DESCRIPTION OF THE INVENTION The present invention provides various embodiments, I i including at least one of: a vector comprising at least one nucleic acid such as DNA for cloning of a nucleic acid or for RxprpRPion of at least one heterologous protein by a cell such as gram negative bacteria {the vector can comprise a nucleic acid molocule, e.g., DNA, encoding: an origin of replication region, optionally and preferably a selection marker (which can be a eodi nq nucleic acid in a restriction site) , a promoter, an i ni i i at ion region e.g. a translation initiation region and/or a ribonome binding site, at least one restriction site for i nsort ion of heterologous nucleic acid, e.g., DNA, encoding the heterologous protein, and a transcription terminator); a method for extracting a recombinant protein from within a cell such as a vncnmhi nnnt Gram negative bacteria having a cell membrane; and, a mothod for purifying an isolated recombinant human insulin. Without limiting the general nature of the foregoing, the following provides a discussion of various embodiments, in detail. In an embodiment, the present invention relates to a method for permeabilization of a cell membrane of a cell such as a recombinant Gram negative bacteria, to extract a protein such an a recombinant protein, from within the cell membrane.' Heterologous proteins are proteins which are normally either not produced by a host cell, or those which are normally produced only in limited amounts. The advent of recombinant DNA I-'■I'hnol.ogy and other standard genetic manipulations, such as point mutagenesis, has enabled the production of heterologous proteins in copious amounts from transfected host cell cultures. In practice, these heterologous proteins are frequently proflnrpf] by genetic expression in quantities that involve pr^ripi tat ion under conditions which maintain the solubility of host, cellular proteins. The present invent ion is directed to procedures for producing heterologous proteins and to methods of isolating and purifying heterologous proteins having minimal contamination by endotoxi ns. The present invention further relates to a method of producing proteins by recombinant DNA technology. The invention relates to: a mvilti -purpose vector far expressing at least one heterologous protein in cells such as E. coli or other gram i n^gntive bacteria; methods for producing such vectors; a method for extracting protein from a cell, such as a recombinant protein i from bacteria, without lysing the cell or bacteria;.and a method for puri fying isolated recombinant protein. Recombinant DNA technology has enabled the expression of foreign (heterologous) proteins in microbial and othei: host colls. In this process, a vector containing genetic material directing a host cell to produce a protein encoded py a portion of a heterologous DNA sequence is introduced into the host, and . i i the transformed host cells can be fermented and subjected to i conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein. Plasmids are extensively used as vectors to clone DNA moi -niinn. Most plasmid vectors 'are made by taking DNA from a v.i ri nfy of repl icons (plasmids, bacteriophage chromosomes and Kir-1 eri.i] chromosomes) and joining the DNA together (using i rr-nt riction enzymes and DNA ligase} to form a plasmid which has p\n origin of replication, a selection marker (usually an an' ibiotic- resistance gene) and a promoter for expressing genes of i ntprest in the required host cell. > In the present invention, DNA encoding a protein such ;=in a prpcursor protei n is inserted into a vector. The coding snqMonro to he expressed is inserted in the correct relationship to n hoot-specific promoter and other transcriptiopal regulatory pfqunnrps! and in the correct reading frame, so that the }] ! I t 1IP plasmid. ; In a preferred embodiment, the expression, vector of the present invention, denoted pLMT8.5, contains the following:1 i. i. Origin of replication, preferably of'pUC8 (which insures a high copy number of the plasmid in the E. coli recipient cells); ii. A marker, preferably a tetracycline resistance marker from plasmid pRP4; iii. A promoter, preferably a PL promoter isolated from harM or i nphage 1 ambda ; iv. Shine-Dalgarno regions, preferably synthetic Shinn-Dalgarno regions, and preferably such synthetic regions from T7 phage gene 10; v. A transcription terminator such as synthetic eCf icient transcription terminator which is Rho-independent; and vi. At least one restriction site, and preferably a region of multiple restriction sites to facilitate the cloning of t.h« rjonfR to be expressed. , ! The construction of the plasmid pLMT8.5 is illustrated in Figs. 1-5, and Figs. 10A-M and 11A-F show the nucleotide sfqiK'nre and restriction sites in pLMTS. 5. Into the at least one restriction site can be cloned at least one nucleotide sequence which can be exogenous, e.g., encoding an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, a fun ion protein or other protein of interest (e.g., 'proinsulin) or combinations thereof. With respect to these terms, reference is mad^ to the following discussion, and generally to Kendrew, THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY (Blackwell Science Ltd., 1995) and Sambrook, Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, 2nd Ed-. Cold Snrina Harbor Laboratory Press, ] 9P2 . An epitope of interest is an immunologically relevant region of an antigen or immunogen or immunologically active fragment thereof, e.g., from a pathogen or toxin of veterinary or human interest. An epitope of interest can be prepared from an antigen of a pathogen or toxin, e.g., an antigen of a human pathogen or toxin, or from another antigen or toxin which elicits a response with respect to the pathogen, or from another a.tigen or toxin wliinh elicits a response with respect to the pathogen, such as, i fnr instance: a Morbillivirus antigen, e.g., a canine distemper virus or measles or rinderpest antigen such as HA or F; a rabies glycoprotein, e.g., rabies glycoprotein G; influenza antigen, °.n., influenza virus HA or N or an avian influenza antigen, i ' --1 no herpesvirus, pseudorabies virus, canine herpesvirus, HSV, Mnrck'R Disease Virus, Epstein-Barr or cytomegalovirus; a flavivirus antigen, e.g., a Japanese encephalitis virus (JEV) antigen, a Yellow Fever antigen, or a Dengue virus antigen; a malaria (Plasmodium) antigen, an immunodeficiency virus antigen, e.g., a feline immunodeficiency virus (FIV) antigen or a simian immunodeficiency virus (SIV) antigen or a human immunodeficiency virus antigen (HIV); a parvovirus antigen, e.g., canine parvovirus; an equine influenza antigen; an poxvirus antigen, e.g., an ectromelia antigen, a canarypox virus antigen or a fowlpox virus antigen; an infectious bursal disease virus antigen, e.g., VP2, VPS, VP4; a Hepatitis virus antigen, e.g., HRRAq; a Hantaan virus antigen; a C. tetani antigen; a mumps antigen; a pneumococcal antigen, e.g., PspA; a Borrelia antigen, i e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli and Borrelia gar in i i; or a chicken pox (varicella zoster) antigein. Of course, the foregoing list is intended as exemplary, nr. rhe epitope of interest can be derived from any antigen of any veterinary or human pathogen; and, to obtain an epitope of interest, one can express an antigen of any veterinary or human pathogen. Nucleic acid molecules encoding epitopes of interest such as those listed can be found in the patent and 'scientific literature such that no undue experimentation is required to practice the claimed invention with respect to any exogenous DNA encoding at least one epitope of interest. Since the heterologous DNA can be for a growth factor or therapeutic gene, reference is made to U.S. Patent No. 5,352,479, which is incorporated herein by reference, together with the documents cited in it and on its face, and to WO 9-1/1^7] fj, each of which is also incorporated herein by reference, toqr-iher with the documents cited therein (see Kendrew, supra, r>npprin"lly at page 4ri'5 et seq. ) . The growth factdr or therapeutic gene, for example, can encode a disease-fighting protein, a molecule for treating cancer, a tumor suppressor, a cytokine, a tumor associated antigen, or interferon; and, the growth factor or therapeutic gene can, for example, be selected from the group consisting of a gene encoding alpha-globin, beta-globin, gamma-globin, granulocyte macrophage-colony stimulating factor, tumor necrosis factor, an interleukin, macrbphage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, mast cell growth factor, tumor suppressor p53, rot i noblnntoma , interferon, melanoma associated ""antigen or B7 . U.S. Patent No. 5,252,479 provides a list of proteins whichcan bo expressed in an adenovirus system for gene therapy, and the skilled artisan is directed to that disclosure. W0: 94/16716 provide genes for cytokines and tumor associated antigens and the I skilled artisan is directed to that disclosure. As to epitopes of interest, one skilled in the art ,can del-ermine an epitope or immunodominant region of a peptide or pn'ypepl ide and ergo the coding DNA therefor from the knowledge op lhe amino acid and corresponding DNA sequences of the 'peptide or polypeptide, as well as from the nature of particular amino ncids (e.g., size, charge, etc.} and the codon dictionary, without undue experimentation. See also Ivan Roitt, Essential Immunology, 1988; Kendrew, supra; Janis Kuby, Immunology (1992), ]->p. 79-80; Bocchia, M. et al. Specific Binding of Leukemia Oncogene?. Fusion Protein Peptides to HLA Class I Molecules. Blood 8^:2680-2684; Englehard, VH, Structure of Peptides associated with class I and class II MHC molecules Ann. Rev'. Immunol. 12r]Rl (1994)); Geffer et al., U.S. Patent No. 5,019,384, issued May 28, 1991, and the documents it cites, incorporated herein by n-f'Tence (Note especially the "Relevant Literature" section of thir? patent, and column 13 of this patent which discloses that: "A large number of epitopes have been defined for a1 wide variety of organioms of interest. Of particular interest aire those ppi l-npps to which neutralizing antibodies are directed. Di ^closures of such epitopes are in many of the references cited i in the RPIevant Literature section.") ! With respect to expression of a biological response modulator, reference is made to Wohlstadter, "Selection Methods," WO 93/19170, published 30 September 1993, and the documents cited therein, incorporated herein by reference. j ! With respect to expression of fusion proteins by : inventive vectors, reference is made to Sambrook, Fritsch, Mnniatis, Molecular Cloning, A LABORATORY MANUAL (2d Edition, rv->ld Spring Harbor Laboratory Press, 1989) (especially Volume 3), and Kendrew, supra, incorporated herein by reference. The teachings of Sambrook et al., can be suitably modified, without undu" experimentation, from this disclosure, for the skilled *.irv \nr\Ti to generate recombinants or vectors expressing fusion pt i >*- r-i ns . Thus, one skilled in the art can create recombinants or vor-t-^rp oppressing a growth factor or therapeutic gene and use j' tho vpromliinflntg or vectors, from this disclosure and the kn^wledge in the art, without undue experimentation. Moreover, from the foregoing and the knowledge in the art', no undue experimentation is required for the skilled "artisan to ctiiiptnict: an inventive vector which expresses ;an epitope of int«rn=it, a biological response modulator, a growth factor, a recognition sequence, a therapeutic gene, or a fusion'protein or any protein of interest such as pro-insulin; or fori the skilled art- i p.an to use an expression product from an inventive vector. Further preferred embodiments of the invention include plnsmid vectors containing a nucleic acid molecule {inserted into a rnRtriction site) encoding pro-insulin and pro-insulin with a HIP tag, e.g., plasmids pPTAl and pHIS (which are akin to pT,MT".5, but contain DNA encoding pro-«insulin and pro-insulin I wiMi a Mi 5? tag in a restriction site; see Figs. 12 and 13). The I ■ pro insulin with a His tag is .useful for isolation of the prb- j i innulin (see Example 7). ; The stability of the protein can be a limiting factor in the expression of its gene in E. coli, which is affected by many factors, including the presence or absence of proteolytic Enzymes in the medium, as well as the sequence of the protein i 1-RPI f . The formation of inclusion bodies of the produced rprombinant protein can facilitate protection ag.iinst proteolysis. The inclusion bodies are produced depending on the protein, and can have certain advantages if one wants to induce protp ins which are insoluble or which are toxic for E. coli (fir-ti^in, 1939; Kane & Hartley, 1988; Hellebust et al. , 1989). ['pnora 1.1 y, they are formed as cytoplasmatic aggregates which can i I if; purified after lesion of the cell followed by centrifugation and mixi ng the proteins with a strong denaturant, e.g. urea or rjnanidinp . With regard to the stability of the induced protein as jt is affected by the protein sequence, the half-life of a pmi-nin should also be considered in relation tp its amino- tfrniinal residue, also known as N-end rule. Tobias et al. (1991) conf i rm the existence of this rule in E. coli. The residues arninine, lysine, leucine, phenylalanine, tyrosine and tryptophan at tin-1 amino terminus, tend to decrease the half-life of the protein, i.e., the half life can be on the order of two minutes, wh^r^as other residues provide proteins having a half-li fe of morr- than ten hours for the same protein. The amino acids arqinine and leucine act as secondary destabilizing residues hfrnnse their activity depends on conjugation with the primary clr-rf abi 1 i ?.\ ng residues, leucine and phenylalanine, through the PATENT 54-0510 2003 t" ransference protein-tRNA-phenylalanine/leucine (transferase r-/F) ■ Thin enzyme, which is present in Gram-negative bacteria and absent in eukaryotea, catalyzes the conjugation of leucine and phenyl alanine in N-arginine ends and lysine sterically accessible in proteins or peptides. The protease Clp(Ti), one of the two known ATP dependent proteases in E. coli (the other one is La), is needed for degradation in vivo of N-end rule Hiibfitrarps. Clp (75 0 kd) is a protein containing two subunits, ripA (?.\ Kda) and ClpP (21 Kda) , being comparable to the 20 S prnteasomes of the eukaryotes. Even though the mutations clpA" of E. coli lose the standard, of the N-end rule, they grow at the rate of the wild E. coli, showing normal phenotypes and not ' stablizing various short-life proteins of E. coli (Varshavsky, 1 09?.) . Another way to form a more stable, heterologous protein in F,. coli in by producing it as a protein that fuses with a part of tint i vf/natural protein of the bacteria. Some of the heterologous proteins are rapidly degenerated by the protease of i tie hnnt and genetic fusion stabilizes the produced protein wi'lnn the cell, also providing a strategy for later purification ':-orwond, 1991). One example of this method is the addition of a region, rich in arginine at the carboxyl-end of urogastrone, that aids in the protection against proteolysis and in the puri f i rat-ion of the protein in an ion-exchange column (Smith et a 7., 19R4) . The present invention provides an alternative method Cor the development of stable, isolatable, heterologous proteins, which method overcomes the above-identified problems associated with the stability of the induced protein. The present invention provides a method to improve the expression of the heterologous proteins, by employing a vector for expression, the plasmid pLMTS.5 and derivatives thereof, e.g., pPTAl and pHIS, which are strong enough to result in a rate of protein production higher than the degradation rate. The present invention provides a process for constructing a vector for expression of heterologous proteins, preferably low molecular weight proteins, e.g., less than 10 Kda, in E. coli, The present invent ion provides a highly efficient process for thermo-regulated production of heterologous proteins in E. coli and in other Gram-negative bacteria, preferably for i h' product ion of human pro-insulin. The method of the present invention for thermo-regulated highly efficient production of heterologous proteins in R. call and other Gram-negative bacteria, includes thermal induction of a culture of bacteria containing the plasmid pLMT8.5 nnd the gene for cloning, in which the plasmid p^LMTB. 5 is prepared according to the process described herein and the cloning in achieved without genetic fusion. In a preferred embodi meat, the heterologous protein is human pro-inaulin from the synthetic gene for pro-insulin. Recombinant E. coli cells almost always express the lie! erologous protein in the form of insoluble cytoplasmic inclusion bodies. In other words, the recombinant protein is not excreted into the culture media. An additional characteristic of recombinant E. coli is the accumulation of high amounts of acetate in the media, mainly'during the induction phase. The deleterious effect of acetate accumulation (>5g/L) on cell growth and recombinant protein expression is well documented in the literature. Additionally, with regard to the accumulation of high concentrations of acetate in the media, which is a general consequence of working with E. coli, the present invention facilitates the development of fermentation conditions, wherein both high biomass accumulation (>70 g/dry weigh.,/L)! and maintenance of low acetate concentration { With regard to the method outlined herein for the purification of protein isolated from inclusion bodies, it will be understood that minor modifications in the purification protocol may be made without departing from the spirit or scope of the invention, i.e., specifically in regard to the choice of solvents, buffers, detergents, denaturants, proteolytic enzymes, separation methods and chromatographic media. It will be understood from the disclosure that while the preferred detergent for une in the method of the present invention is Triton X-100, one of ordinary skill in the art may employ any such nonionic detergent in practicing the instant invention. Similarly, with regard to the choice of proteolytic enzymes, while trypsin and carboxypeptidase B are preferred, one may substitute any appropriate proteolytic enzyme, e.g., the substitution of FYidoproteinase Lys-C for trypsin, in order to convert pro-insulin to insulin, and such a substitution is well within the gambit of knowledge of the skilled artisan acquainted with available proteolytic enzyme preparations and their respective specificities. A better understanding of the present invention and of its many advantages will be had from the following non-limiting Examples, given by way of illustration. EXAMPLES Example 1 - Vector Preparation The inventive process for the construction of a vector for use in thermo-regulated production of heterologous proteins in E coli, and the construction of an inventive vector of the invention was comprised of the following stages: i. Construction of plasmid pULTDK7.1 (Figure 1) The construction of pULTDK7.1 was initiated by the isoJation of the fragment containing the promoter PL of the phage lambda. This fragment extends from the HindllJ site to the Hpal site of the phage and was cloned into the Hindlll and Smal sites of the polylinker of pUC19, forming the recombinant plasmid pUCPL2.7. Oligonucleotides 011929 and 011930, which contain the Shine-Dalgarno region of the gene 10 of the phage T7, were i annealed and ligated to the EcdRI site of pUCPL2.7, forming ; ':' : ■ plasmid pULT7.2.4. Plasmid pULT7.2.4 was cleaved'at■the BspEI i and Xbal sites, treated with DNA polymerase I fragment Klenow and relegated, resulting in a deletion of the coding region of the gene N of the phage lambda and formation of plasmid pULTDK7.1. ii. Construction of Plasmid pUTC6 (Figure 2) The construction of pUTC6 began by the isolation of the gene for resistance to tetracycline (Tc), by means of digestion of the plasmid pRP4 with Bglll and StuI, which liberated a fragment of 1.4 kb, which was cloned in the BamHI and Smal sites of plasmid pUC8, forming plasmid pUTl., A 0.9 kb pU^R fragment was liberated by digestion with Oral and PvuII and lighted to pUTl after digestion with PstI, treatment with SI nuclease, digested with EcoRI and treated with Klenow, to obtain the plasmid pUTC6, which maintains the site EcoRI and contains the origin of replication and the gene for resistance to Tc. iii- Construction of Plasmid PLMC8.1 (Figure 3) Annealed oligonucleotides 1 and 2 were ligated to the BcoRI site of pUTC6 to form the plasmid pMC8, containing a region of restriction sites for molecular cloning., Subsequently, pULiTDK7 .1 was liberated by digestion j with ;■ the Hindlll and BcoRI and ligated to pMC8, also digested with Hindi 11 and BcoRI, to yield the recombinant ple_3mid pLMC8.1, containing the gene for resistance to Tc, the origin of. replication, the promoter PM the Shine-Dalgarno region and a polylinker for molecular cloning. iv. Construction of Plasmid pLMTB . 5' (Figure 4) An efficient transcription-terminator was inserted from oligonucleotides 12 and 13 which were annealed and ligated to the BamHl site of the plasmid pLMCS.l, preserving the site at the 5' end of the oligonucleotides and creating a Hindlll site at the 3' end, yielding the expression vector pLMTS.5 for E. coli and other gram-negative bacteria. {SEQ ID NOS: ). Hence, pLMTS.5, prepared according to the method | described hereinabove, contains: (1) origin of replication of i pUCG; (2) the gene for resistance to tetracycline of pRP4; (3) i ' 1 IIP promoter PI of the Lambda phage; (4) the SD-region of the gene 10 of the phage T7; (5) multiple cloning sites for cloning without fusion; and (6) Rho-independent terminator of t ranscription (See Figs. 1-5, 10A-M, 11A-F) . , > To obtain a vector expressing pro-insulin, coding DNA therefor was inserted into a restriction site of the multiple cloning sites of vector plasmid pLMT8.5. In particular, the synthetic gene for proinsulin was cloned on a polylinker yielding vector plasmid pPTAl (pLMTS.5 + proinsulin). The vector was then inserted into E. coli and cultures thereof were grown for expression of pro-insulin (e.g., N4830-1 (cl857) strain; see Examples below). v- Construction o£ plaamids pLA7 and pHIS A fragment from pLA7 containing the pro-insulin sequence and a histidine tag was inserted into the restriction site of the multiple cloning sites of vector plasmid pLMT8.5 yielding vector plasmid pHIS. The cloning strategy is depicted in Figures 12 and 13. The vector was then inserted into E. coli ^nci cultures thereof were grown for expression of pro-insulin (e.g., N4830-1 (cla57) strain; see Examples below).1 pHIS contains the pro-insulin gene with the oligo encoding a (HIS)6 insertion (Met-Ala-His-His-His-His-His-His-Met-Gly-Arg). (The synthetic gene f6r proinsulin, constructed by .i riven tors, using ol igonucleo tides was, cloned into the Ncol and a? mil I sites in the polyl inker region of pLMT8.5 vector, to make the pPTAl and pHIS proinsulin expression vector, see Example 8.) Example 2 - High Biomaas Formation with Low Acetate Accumulation Experimental results showed that programmed additions of yeast extract were necessary for high biomass formation. To maintain the acetate concentration at low levels, the pH of the fermentation was controlled automatically via a glucose loop. Under these conditions not only could the pH be controlled at any desirable value (e.g. 6.8 or lower), but also the glucose level in the fermentation broth could be maintained at very low levels ( accumulation of acetate. Under these conditions dry cell weights of up to 95g/L and expression of l94mg of fusion protein per gram of dry cell weight were obtained. Example 3 - Penneabilization and Solubilization of Inclusion Bodiea Containing Pro-Insulin ___ As a rule, inclusion bodies are recovered from the host organism by two procedures: (a) Mechanical or physical rupture of the cell envelope' by passing the cell paste through a Manton-Gaulin press or by grinding the cell suspension in a colloidal mill such as a Dyno-Mill; and (b) Digestion of the cell envelope by treatment with lysozyme. However, both of the above-identified techniques are costly, and potentially. detrimental to the overall yield of the desired protein. Hence, the method of the present invention employs i i alternative methods for the purification and direct solubilization of inclusion bodies. B. coli cells (K-12 strain W 4830-1 containing the pro insulin gene under the control of the PL promoter; plasmids pPTAl rind pHTS, see Example 1) were grown in 10 liters of medium r-f.ritaJ.ning yeast extract (20 g/L), peptone (6.0 g/L), NaCl (5.0 g/L), glucose (10.0 g/L), Ant i foam A (2.0 g/L), and ampicillin (10') uq/ml ) , pH 6.8 (after sterilization). The medium was iNor-ula^*] at. 10% volume with a pre-culture prepared from an i.^'lriiod colony grown overnight in the same medium. At an opt i ':,i I density of approximately 6.0 at 540 nm, synthesis of pro insulin was induced by raining the temperature from 30 to in°c. Induction could alno be initiated at temperatures of 40 to 12 °C. Cells were harvested after 2 to 5 hours of induction. Inclusion body formation was monitored by phase contrast mi rroscopy. The celln were harvested by centrifugation, f~>nuupended twice in de.ionized water, and recovered by '.•rut ri f ugat ion. Known amounts of wet cell cake were resuspended in 0.1M Tris-Hcl, pH 8.5, and appropriate amounts of prrmnabi 1 i ?.at ion compounds, alone, or in combination, were added to n concentration of up to ten times the volume of the weight of )he wet cell cake, as shown in Table 1. After overnight animation at room temperature, the cells were recovered by CMU rifuqation, wet weight of the cell cakes and the wet pellet wore homogenized in 10 times'their weight of 0.1M Tris-Hcl, pH B.5 containing 8M urea, and agitation was continued at room temperature for up to 24 hours. Supernatants were recovered by cmitrifugat ion, and cell pellets were washed by centrifugation. Fusion protein concentrations were determined by SDS-PAGE analynir. using both the supernatant and pellets after the washing nt'^p. Weight determinations made on the wet pellets piior to the pretreatment step, after pretreatment, and after 8M urea treatment showed that a substantial weight loss took place, ar. i=tiown in Table 1. Tab! e .1 - Effect of some pre-treatments on the weight loss of the cell pellets Example 4 - Concomitant Permeabilization and Solubilization of Inclusion Bodies Containing Pro-Insulin In preliminary experiments, the pre-treated cells were cleaned of the cytoplasmic proteins and other contaminating mnt-erial oxtracted from the cells (grown as in Example 3), by rvifilri f uqal i on, foil owed by re suspension of the pre-treated and washed r-M I s in huf fer containing 8M urea. One liter aliquots of a fermentation broth, prepared as i n Example 3, were concentrated to 100 ml by cross flow f i 1 r-ration. Aliquots of the concentrated cell suspension were d i -if i 1 tered with 10 volumes of 0 . IM Tris-Hcl, pH 8.5 buffer roniMininq either 5 Mm EDTA, 1% toluene, or deionized water. r-iid uroa was added to a final concentration of 8M, and the volume war-: brought to 200ml with buffer. Samples taken at .lifforcnt time intervals, up to 24 hours, were analyzed by SDS- PAHR. A highly effective purification and solubilization of the i IK: I uFs i on bodies was obtained in as little as 6 hours of urea f7 Example 5 - Cell permeabilization'procedure using 20% Triton X-100 Cell cultures grown as in Example 4 were harvested by cr>nt r i fugnt ion, re suspended twice in deionized water and recovered by centrifugation. Known amounts of the wet cell cake wro resufipended in 0. IM Tris-Hcl, pH 8.5, containing 20% Triton X -100, and the solutions were agitated overnight at room t emperature. Fusion protein concentrations were determined by SP^-PAHF analysis, and it was found that under these conditions, n oubfitantia I amount of cytoplasmic material diffuses out of the¬re |1, leaving an empty shell containing essentially the inclusion body with few contaminating cellular proteins. Example 6 - Purification and Concentration of the Solubilized Inclusion Bodies by pH Precipitation Cell cultures are grown and pre-treated, and the j rir■ 1union bodies solubilized as in Examples 3 or 4. The solution of r-soliihi 1. i zed inclusion bodies was dialyzed, or ultraf iltered. to eliminate urea. A fractional pH precipitation step resulted in i-l-ip enhanced purity of the solubilized protein. The pH of the pro!ojn solution was lowered, either by the addition of mineral afidrs, i.e., hydrochloride or sulfuric acids, or by organic aritln, i.e., acetic acid. The pH was lowered to 6.0 by this method, and the precipitate was removed by centrifugation. The funion protein was precipitated from the solution by^ lowering the pH to 5.0 by the same method. A complete recovery of the fusion protein was achieved. The precipitated fusion protein was dissolved in alkaline buffer at pH 8.5. The purification of protein by fractional pH precipitation is shown in. Figure 7. Example 7 - Purification and Isolation of Insulin The isolated inclusion bodies or whole pre-treated cells from cells containing and expressing plasmid pHIS were wnnhed twice with 50 Mm ammonium acetate buffer, pH 9.0 and centrifuged. The precipitate was dissolved in 50 Mm ammonium acn|-ate buffer, pH 9.0, containing 8M urea, and sodium sulphite {\ .2% g/q of sample) and sodium tetrathioate (0.55 g/g of sample) were added. The sample was stirred, at room temperature. The sulfitolysis reaction was monitored by analysis of aliquots on a M-.rio-Q column. After 24 to 48 hours, the sample was diluted 3 tinms with deionized water, centrifuged, and the supernatant was filtered to give a clear solution. The filtered supernatant was applied to a Ni-chelating sepharose FF column {Pharmacia Biotech, Upsala Sweden^. equilibrated with 0.1M sodium phosphate, 50mM NaCl, pH 7.3. The sample was eluted in a stepwise gradient, with the equilibration buffer containing 8M urea and 0.08M imidazole, followed by wanning with the equilibration buffer containing- 8M urea and 0. 3M imidazole. The chromatographic separation was monitored by ■ absorbance measurement at 280 nm. The buffer of the solution containing pure S-sulfonated protein isolated by metal affinity chromatography was changed to 10 Mm glycine, pH 10.0, by gel filtration chromatography on Sephadex G-25. The pure sulfonated protein was renatured (0.5mg prniein/ml) by the addition of 0.5mM cystine and 0.5mM beta-m«rcaptoethanol, with agitation for 18 to 24 hours at 4 to 8oc, .'iivl thn reaction was monitored by injection of aliquots of the reaction mixture on HPLC equipped with an Aquapore RP-300 column. The renatured samples were concentrated and the buffer was changed by diafiltration. To 1.0 ml of renatured sample (8 mg/ml) in 0.1M Tris-Hcl, pH 7.5, containing 0.01M EDTA,'was added 35 ug of trypsin and o.^ug of carboxypeptidase B. The reaction was monitored by HPLC analysis on an Aquapore RP-300 column, and the reaction was complete after 1 hour at 37oC. The reaction mixture was diluted 3 times with water, and purified by ion-exchange chromatography and reversed-phase HPLC. Example 8 - Teat of Efficiency of the Vector of Expression A synthetic gene for proinsulin, constructed by invnntors, using oligonucleotides was cloned into the Ncol and RimMf sites in the polylinker region of pLMT8.5 vector to make I tic pPTAI and pHIS proinsulin expression vectors (see Example 1). In l-psts it was found that, after thermal induction of a culture of R. cali N4930-1 (clflS7) strain,1 containing the plasmid pPTAI (pIiMT8.5 + proinsulin), there was induction of the recombinant prntein of approximately 10 Kda, in a fraction of 20% of the tninl proteins of the bacteria over a period of 90 minutes, and tli.it it loses the capacity to multiply during the thermal shock, leaving it only to the production of the recombinant protein, as nhown in Fiquire 4. The plasmid pPLT4 was also used in this test. This plnsmid is a derivative of the plasmid pPLc28 (Remaut et a"!., 1981), modified by inventors, in which the same synthetic Rhiiip Dalgarno site and the proinsulin gene of the plasmids pT.MTR.1; mid pPTAI were cloned. By comparison with this plasmid, nh"w i nq -in induction of the protein of 11% and cell-growth during t h" i hernial shock, it was found that, over a period of 90 iiiiiiut-ps, p!,MT8.5 was 100% more efficient (See Fig. 9). In this manner a hyper-expression-vector, for E. coli, denoted pI>MT8.5, was obtained. When tested on the production of human proinsulin from the synthetic gene, high levels of protein PxproRRinn were found to be induced from this gene. Plasmids pI,MT8 . 5, pPLT4, pHIS, and pPTAl were deposited on June 24, 1997 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, USA, under ATCC accession numbers 98474, 98475, 98476 and 98473. Example 9 - Additional Expression Vectors A gene for any of: an epitope of interest, a biological response modulator, a growth factor, a recognition sequence, a trlierapent ic gene, a fusion protein or another protein of interest or combinations thereof (as discussed in the Detailed Description) cloned on a polylinker and inserted into plasmid of Example 1, e.g., pLMTS.5, pHIS, and pPTAl; and, plasmids resulting therefrom are inserted into E. coli, e.g., N4830-1 (el"") strain (containing the plasmid pLMT8.5 + gene). After t henna] i nduct ion rhere is induction of the recombinant protein aki n to that observed in Example 8 showing that pLMT8.5 is extremely efficient. In this manner a hyper-expression-vector for E. coli, denoted pLMT'8.5, is obtained. When tested on the production of human proinsulin from the synthetic gene, high levels of protein rxprespi on were found to be induced from this gene; and, high ]»VP!S of expression are obtainable from using vector plasmid pl.M'l'B . s ?\i\d other exogenous genes. As discussed herein, the -^lrcL ion marker can be omitted from pLMT8.5 or derivatives thereof, e.g., pHIS, and pPTAl, and selection can be based on expression of a gene product, e.g., of insulin or of insulin with n (MR tag. Methods for selection based on expression of an exogenous coding sequence are known in the art and can include immunoprnripitation or other antibody-based screening methods which employ antibodies which bind to the expression product, or r-nli'ct-ive me,dia with respect to the expression product (see, e.g., U.S. Patent Nos. 4,769,330, 4,603,112, 5,110,587 regarding Rejection using selective media with respect to an expression product; U.S. Patent No. 5,494,807 regarding selection using ant- i body-based screen ing methods) . Example 10 - Manipulation of fermentation conditions to enhance protein expression Productivity of the fermenter could be increased PubRtantially (C for an additional 5 hours, followed by an additional 5 hoiirs of induction at 42»C. In this way, without increasing the overall fermentation time (20-22 hrs.), an increased volume of biomass and recombinant protein is obtained. Thus, alternating 5 hours of fermentation at 30°C with 5 hours of induction at 42°C, resulted in a higher percentage of recombinant protein expression than when starting the induction nft-er prolonged fermentation (approximately 17 hours) at 30°C. Heat inactivation was found to negatively influence the iiiflusion body pur if i oati on steps due to considerable coagulation of oyt oplasmic proteins, at the heat inactivation temperature Pel 1 inact ivation and permeabilization was performed mnconiiinnfly by overnight treatment of the harvested cells with 1 9; Toluene and 50 Mm EDTA. -Further purification was achieved by r".".unpendi Tig the recovered biomasa in Tria 0 . 1M, pH 8 . 5 buf Eer, rout T in.i ng 1.0% Tri ton X-100 , and agitating for five hours or OVPI n ight . Cel 1 s pre-treated in this manner, after cent ij f ugat- ion, can be used directly in the ensuing puri f ication fit °| >S . Additionally, by further lysozyme treatment, a pn. Having thus described in detail preferred embodiments of t he present invention, it is to be understood that the invention defined by the appended claims is not to be limited to part i on]ar details set forth in the above description as many apparent vari at ions thereof are possible without departing from ihe spirit or scope of the present invention. REFERENCES 1. Denhardt, D.T. & Colaaanti, J. 1987. A survey of vpftors for regulating expression of cloned DNA in E.coli. In VW-tors - a survey of molecular cloning vectors and their uses. R.r,. Rodriques and D.T. Denhardt, eds. Butterworth Publishers, Soneham, MA, U.S.A. 2. Remaut, E. ; Stanssens, P. & Fiers, W. 1981. Plasmid vp'lors for high efficiency expression controlled by the PI promoter coliphage ]ambda. Gene, i5 :81-93 . 3. Mellado R.P. & Salas, M. 1982. High level synthesis in I-'nrhnrichia coli of the Bacillus subtilis phage \i'i -itiff p'l under the control of phage lambda PL promoter. NU'I .Acid.s Res., i0.:5773-84. ■1 . Reman 11 E. ; Stanssens, P. & Fiers, W. 198 3a. I n'iiK-i ble high level synthesis of mature human fibroblast i tit f-rf eron in Escherichia coli . Nucl. Acids Res . , H:4677-88 . 5. Simons, G.; Remaut, E.; Allet, B. Devos, R. & Fiers, W. 1984. High-level expression of human interferon gamma in Escherichia coli under control of the PI promoter cf ba 6. Remaut, E.; Tsao, H. & Fiers, W. 1983b. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication . Gene, 2.2.: 103 - 13 . 7. Crowl, R. ; Seamana, C; Lomedico, P. & McAndrew, s. 1 0Rr;. Versatible expression vectors for high-level synthesis of cl oned gene products in Escherichia coli . Gene, 3J3: 31-8 . 8. Lautenberg, J.A.; Court, D. & Papas, T.S. 1983 . Hiqh level expression in Escherichia coli of the' carboxy-terminal R^qunnceR of the avian myelocytomatosis virus (MC2 9) v-myc pi-n( n i n . Gene , 23 : 75 -84 . 9. Seth A.; Lapis, P.; Vande Woude, G.-F. & Papas, T. 1 ')xr, . Hi qh level expression vectors to synthesize unf used pintninR in Escherichia coli. Gene, 42:49-57. 10. Cheng X. & Patterson, T.A. 1992. Construction and ']"'■' of I PL promoter vector for direct cloning and high level fxprpfision of PCR amplified DNA coding sequences. Nucl. Acids R^., 20M591-8. 11. Schauder, B. ; Blocker, H. ; Frank, R. & McCarthy, i'.R.O. 19(17. inducible expression vectors incorporating the F.r.rhnrichin coli atp E transl at ional initiation region. Gene, F:> :'M0- 8 3 . 12. Rosenberg, M.; HO, Y.S & Shatzman, A. 1983. The ii!"5 of pKC3 0 and its derivatives for controlled expression of q^nes. Mnth. in Enzymol., 101 : 123-38. 13. Chaconas, G. ; Gloor,G'. & Miller, J.L., 1985. Amplification and purification of the bacteriophage Mu encoded B t- rnnnposition protein. J . Biol. Chem. , 260:2662-9. 3 4. Lowman, H.B.; Behm, M.; Brown, S. & Bina, M. 1988. Hi gh-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion protein in Escherichia coli . Gene; 6,8 : 2 3 - 3 3 . 15. Mott, J.E.; Grant, R.A.;H0, Y.S. & Piatt, T. 1985. Max i mi zing gene expression from plasmid vectors containing the X ri, promoter : strategies for overproducing transcription i frmi nat- i on factor p . Proc .Nntl . Acad . Sci-USA, 82.: 88-92 . 16. Schein, C.H. 1989. Production of soluble i nr^niibi nant proteins in bacteria. Bio/technology, l_:1141-9. 17. Kane, J.F. & Hartley, D.L. 1988 . Formation of r.'"'nmbinant; protein inclusion bodies in Escherichia coli. Tihfnch, 6:95-101. 18. Hellebust, H.; Abrahmsen, L.; Uhlen, M. & Enfors, P.O. 1995. Different approaches to stabilize a' recombinant fusion protojn. 11 io/technol ogy, 2:165-8. 19. Tobias, J.W.; Shrader, T.E.; Rocap,- G. & Vnrahnv.'iky, A. 1991. The N end rule in bacteria. Science, 2r?4: 1371-7. 20. Varshavskiy, A. 1992. The N-end rule. Cell, 69:725-3 r-.. 21. Smith, J.C.; Derbyshire, R.B.; Cook, E.; Dunl.horno, L. ; Viney, J. ; Brewer, S. J-. ; Sassenfeld, H.M. & Bell, L.n., 1984. Chemical synthesis and cloning of a poly (arginine)-mdinq gene fragment designed to aid polypeptide purification. '■CUP, 32:321-7. WE CLAIM: 1. A method for extracting a recombinant non-membranous protein including proinsulin from within inclusion bodies of a recombinant Gram negative bacteria having a cell membrane, without lysing the bacteria comprising the steps of; (a) permeabilizing the cell membrane by contacting the bacteria with a detergent such as herein described to separate native cell proteins from the cell membrane without separating the recombinant protein from the cell membrane; (b) sol ubilizing the recombinant protein and the cell membrane in a known manner, and (c) separating the recombinant protein from the cell membrane in a known manner. 2. The method as claimed in claim 1, wherein in step (a) the detergent is Triton X-100. 3. The method as claimed in claim 1, wherein in step (a), the native cell proteins are separated from the cell membrane by centrifijgation and a resultant pellet therefrom contains the recombinant protein within the cell membrane. 4. The method as claimed in claim 1, wherein in step (b), the recombinant protein is solubilized by contacting the recombinant protein within the cell membrane with urea. 5. The method as claimed in claim 1, wherein in step (c), the separating is by centrifugation and recovering said recombinant protein from a resultant supernatant. 6. A method for extracting a recombinant non-membranous protein including proinsulin substantially as herein described with reference to the accompanying drawings.

Documents:

1479-mas-1998 abstract.pdf

1479-mas-1998 claims.pdf

1479-mas-1998 correspondence-others.pdf

1479-mas-1998 correspondence-po.pdf

1479-mas-1998 description (complete).pdf

1479-mas-1998 drawings.pdf

1479-mas-1998 form-2.pdf

1479-mas-1998 form-26.pdf

1479-mas-1998 form-4.pdf

1479-mas-1998 form-6.pdf

1479-mas-1998 petition.pdf