Refolding Proteins from E. Coli

Refolding Proteins from E. Coli -...

Info iconThis preview shows pages 1–5. Sign up to view the full content.

View Full Document Right Arrow Icon
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 2
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Background image of page 4
Background image of page 5
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: -mv—~—----~-~»body-proteinsis~a—field--of»increasinginterestiongaining. large VVVVVVV Wresulting,in_aggregation.7_ l p i v‘» t /. I 497 (3 Advances in reioiding of proteins produced in E. coir" “ H'auke Lilie, Elisabeth Schwarz and Rainer Rudolphi‘ 0 Inclusion body production is a common theme in recombinant protein technology. Hence, renaturation of these inclusion amounts of proteins. Recent developments of renaturation procedures include the inhibition of aggregation during refolding by the application of low molecular Weight additives and matrix-bound renaturation techniques. Addresses Instituter Biotechnologie, Martin Luther Universitat HalIe-Wittenberg, Kurt Mothes Strasse 3, D-06120 Halle, Germany *e-mail: rudolph@biochemtech.uni-halle.de ' Current Opinion in Biotechnology 1998, 91497—501 ' http://biomednet.com/elecref/O958166900900497 © Current Biology Ltd ISSN 0958-1669 Abbreviation deCl guanidinium hydrochloride introduction Elegant and well established recombinant DNA method- ologies have set the Stage for the production of heterologous proteins in microbial hosts. The abundance of protein expression systems renders the efficient bacter— ial production of most proteins possible; however, high level expression of recombinant procein often results in aggregation and accumulation in inclusion bodies. Deposition of the recombinant protein in inclusion bodies can be heaven or hell. The latter applies to those proteins for which renaturation is problematical. Here, the only way out is the avoidance or at leaSt reduction of inclusion body formation. In contrast, in the case of a simple and efficient renaturation procedure, deposition of the protein in inclu- sion bodies and subsequent isolation and renaturation of inclusion body protein often means the most straightfor- ward strategy to get large amounts of the recombinant protein. In this review we present an extract of recent developments in inclusion body refolding. Inclusion body formation Upon overexpression of recombinant proteins, inclusion bodies can be observed in several host systems, for exam- ple, prokaryotes, yeast or higher eukaryores. Even endogenous preteins, if overexpressed, can accumulate in inclusion bodies [1], suggesting that in most cases inclu- sion body formation is a consequence of high expression rates, regardless of the system or protein used. There is no direct correlation between the propensity of inclusion body formation of a certain protein and its intrinsic prop- erties, such as molecular weight, hydrophobicity, folding pathways, and so on. Only in the case of disulfide bonded proteins can inclusion body formation be anticipated if the protein is produced in the bacterial cytosol, as formation of disulflde bonds does usually not‘ occur in this reducing cel— lular compartment. The consequence is improper folding An increase in the concentration of non-native polypep- tides tlue to high expression levels seems to be responsible for aggregation of the recombinant protein. This assump— tion was quantified in a kinetic model that analysed the yield of native protein as a function of the competition between folding and aggregation [2]. According to this model, the relative yield of native protein increased with a decreased rate of protein synthesis. Qualitatively, this was confirmed by recombinant protein expression at optimal and suboptimal conditions. Thus, whereas recombinant proteins often aggregate when Err/mrit/Iizl coli cells are cul- tivated at 37°C, redLiCtion of the cultivation temperature can increase the amount of native protein due to a decrease of the rate of protein synthesis [3]. Alternatively, the addition of non—metabolizable carbon sources, such as desoxyglucose, at the time of induction leads to a reduced metabolic rate, which results in a limit- ed production of the recombinant protein. Another possibility is limited induction of gene expression by the promotors, which can be linearin regulated by the induc- er concentration. Recombinant protein deposition in inclusion bodies is commonly observed with hydrophobic proteins. Here, fusion with a hydrophilic protein can enhance solubility. Widely used fusion partner proteins are glutathione—S—transferase, maltose—binding protein or thioredoxin [4,5]. A way of taking advantage of the host cell’s equipment to deal with protein folding is the co-overexpression of the recombinant protein with molecular chaperones [6,7',8,9]. Still, the beneficial effect of co-overexpression of chaper- one proteins is unpredictable, as the appropriate substrate-chaperone combination is a matter of trial and error [10]. In the case of disulfide-containing proteins, the principles mentioned above are not sufficient to avoid inclusion body formation. Although the reducing conditions of the bacte- rial cytosol allow disulfide bond formation in a few cases [11], disulfides are not normally formed in this compart— ment. In contrast, the prokaryotic periplasm provides oxidizing conditions for disulfide bonding; however, inclu- sion body formation can also occur in the periplasm [12]. In particular, the native periplasmic expression of proteins that possess several disulfide—bonds is problematical as the endogenous periplasmic enzyme disulfide bond oxidore- ductase (DsbA) merely introduces disulfides into proteins, but does not catalyze disulfide reshuffling. A means to enhance' correction of incorrect disulfide-bonds in the ._..-...._.,_‘§W n 498 Expression vectors and delivery systems E. coli expressing the heavy chain of the antibody MAK 33. The cells _ were harvested six hours after induction of expression. Preparation for electronmicroscopy included fixation with glutaraldehydeI embedding in Epon resin and negative staining with uranyl acetate. The inclusion bodies are visible as amorphous light grey structures. ____________________.___————-———-—— periplasm of E. 5011' is to overex'press the endogenous periplasmic Dst protein, which is a disulfide isomerase [13“,14]. Also, cultivation in the presence of thiol reagents, which lead to reshuffling of incorrect disulfide bridges, has been proven to enhance the yield of native proteins con- taining multiple disulfide bridges [15,P1]. Properties of inclusion bodies Inclusion bodies are very dense particles of aggregated protein (Figure 1). Because of their refractile property they can be visualized by light microscopy. Inclusion bodies may reach sizes with a diameter in the umg range and exhibit an amorphous or paracrystalline structure indepen- dent of their subcellular location. Under appropriate conditions the recombinant protein deposited in inclusion bodies amounts to about 50% or more of the total cell pro- tein. These inclusion bodies .often contain almost exclusively the overexpressed protein. Major contaminants of inclusion body material after preparation are outer mem- brane proteins, which are themselves not part of the inclusion body particles but copurify as non-solubilized protein with the inclusion body fraction. Separation of these membrane proteins from inclusion body material can be achieved by extensive washing with detergents. Little is known about the structural properties of the aggregated protein. Structural analyses of inclusion body proteins indicate that the aggregates possess a certain amount of secondary structure [16], a result also observed for in vitro aggregated proteins [17]. Being aggregated polypeptides, inclusion bodies are gener- ally not very sensitive to proteolytic breakdown. Still, degradation products can often be detected in inclusion bodies. After analysis of the expression kinetics, an optimized fermentation protocol can decrease degradation of the recombinant product. A shift to elevated tempera- tures (42°C) at the time of induction to accelerate the aggregation process further decreases proteolysis. Preparation and solubilization ,__.The‘,basic pringiple of inclusion body preparation is a liq- uid/solid separation. Because inclusionmbofldies have a relatively high density (~1.3 mg/ml [18]) they can be pel- leted by centrifugation. It is important that cell lysis is complete, because intact cells sediment together with the inclusion bodies, thus, contaminating the preparation with host protein. The most effective procedure for complete cell disruption is a high pressure dispersion following a lysozyme treatment. A further treatment of the crude lysate with detergents solubilizes lipids and membrane prOteins (for protocol see [l9']). In a few cases, urea or guanidinium hydrochloride (deCl) at low concentrations can dissociate inclusion body associated preteins. It should be kept in mind, however, that a too high urea or deCl concentration will lead to the solubilization of the inclu- sion bodies themselves. On average, an inclusion body preparation contains more than 50% of the recombinant protein, the purity of the preparation may reach up to 90% under optimal conditions. Before starting renaturation the inclusion body material has to be solubilized by strong denaturants, such as 6 M deC1 or 8 M urea. The addition of dithiothreitol keeps all cysteines in the reduced state and cleaves disulfide bonds formed during preparation. Before renaturation dial- ysis againsr deCl at low pH without reducing agents can be performed to remove the dithiothreitol for subsequent storage (for protocol see [l9']). Renaturation of inclusion body proteins Renaturation of solubilized inclusion bodies is initiated by , the removal of the denaturant either by dialysis or dilution. As discussed for the 1'11 viva situation, the efficiency of renaturation depends on the competition between correct folding and aggregation (Figure 2). Aggregation of the non- native recombinant protein may be enhanced by other proteins contaminating the inclusion body preparation if they themselves tend to aggregate [20']. To slow down the aggregation process refolding is usually performed at low protein concentrations, in a range of 10—100 ug/ml. Furthermore, renaturation conditions must be carefully optimized regarding external parameters, such as temperature, pH or ionic strength. Even in an optimized system, however, the yield of renaturation may be relative- ly low, necessitating large volumes for preparation of large quantities of the native protein. Because during refolding only the concentration of non-native polypeptides and not that of native protein is critical for aggregation, a strategy to circumvent the problem of large refolding vessels is pulse renaturation [21]. In order to keep the concentration of the unfolded protein low, thus limiting aggregation, O T. (D I: -¢ 0 N 1 l Advances in refolding of proteins produced in E. coli Lilie, Schwarz and Rudolph 499 glutathione [P2]. The advantage of this procedure is that the mixed disulfide form of the protein carries additional charged residues provided by the glutathione moiety, which increases the solubility of the protein during refolding. Recent studies on disulfide bond formation within pep- : Z .9 .g E 50 50 ‘g‘ 3 E 0 on in cn o: < 0 A 0 0 0.01 CLDH (FM) - |_______________.__4 Effect of protein concentration on the renaturation of lactate dehydrogenase (taken from [18]). Acid denatured lactate dehydrogenase was renatured in 0.1 M phosphate buffer pH 7, 1 mM EDTA, 1 mM DTE. 20°C. After 192 hours reactivation (O) and aggregation (A) were quantified. _____________________—————-———-——- aliquots of denatured protein are added at defined time points to the renaturation buffer. The time interval between two pulses has to be optimized according to the refolding and aggregation behaviour of the respective pro- tein. The pulse renaturation is stopped when the concentration of denaturant, introduced in the refolding buffer together with the denatured protein, reaches a crit- ical level, that is, a concentration at which even the native protein develops the propensity to aggregate. If proteins contain disulfide bonds, the renaturation buffer has to be supplemented with a redox system. The addition ofa mixture of the reduced and oxidized forms of low mol- ecular weight thiol reagents, such as glutathione, cysteine and cysteamine (molar ratios of reduced to oxidized com- pounds 111 to 5:1, respectively), usually provides the appropriate redox porcntial to allow formation and reshuf- fling of disulfides [22,23]. The redox system} described above is characterized by overall reducing conditions. Correct disulfides are proteCted by the stable native struc- ture; however, these correct disulfide bonds within the native structure which are still solvent accessible could be reduced under these conditions. In such a case, a subse- quent strongly oxidizing step using an excess of oxidized thiol reagents [24] or copper-induced air oxidation can ensure formation of the respective disulfide bonds. For certain proteins, probably due to low solubility of fold- ing intermediates, oxidative refolding is not very effective. In this case, the yield of renaturation may be increased if the denatured protein is first completely oxidized in the pres- ence of a large excess of oxidized glutathione leading to the conversion of all SH- groups to mixed disulfides. Renaturation of the protein is performed by dilution in a renaturation buffer containing catalytic amounts of reduced tides showedwthmpossibilitywoLamdirectedwwreactior1. Oxidation of peptides containing both two selenocysreines and two cysteines leads to the formation of two disulfides: one between the selenocysteine residues and another between the cysteines. No disufide bond between seleno- cysteine and cysteine was observed [25]. This result was independent of the order of selenocysteines and cySteines within the peptide. Because the peptide did not possess any conformational constraints directing disulfide bond formation, these results clearly demonstrate the specific reactivity due to the different chemical properties of selenocysteines and cysteines. Whether such disulfide engineering can be transposed on proteins to prevent the formation of wrong disulfides remains to be seen. In addition to the control of parameters such as tempera- ture, pH or redox conditions, the presence of low molecular weight compounds in the renaturation buffer may prove to have a tremendous effect on the yield of _ renaturation [26,27]. Specific cofactors, such as an+ or Ca3+, can stabilize proteins already at the level of folding intermediates, thus, preventing off-pathway reactions. Besides such cofactors and prosthetic groups, a large series of low molecular weight additives are, in certain cases, very efficient folding enhancers: non-denaturing concentrations of chaotrophs, such as urea or deCI, for example, are essential for the renaturation of reduced chymotrypsino— gen A and have been shown to promote folding of several other prOteins [P3]. On the Other hand, by slowing down the refolding kinetics, deCl and urea can shift the com- petition between renaturation and aggregation towards the aggregation reaction. A popular additive is L-arginine [28—30]. In the case of a truncated form of plasminogen aetivator, the yield of renat— uration is about 80% in the presence of 0.5 M L—arginine, whereas in the absence of this additive almost no reactivi- ty was observed [P2]. The mechanism by which L-arginine supports renaturation is Still unknown. L—arginine slightly destabilizes proteins [31] in a manner comparable to low concentrations of chaotrophs. The benefical effect of L-arginine on refolding, however, probably originates from an increased solubilization of folding intermediates. Likewise, increased solubilization of folding intermediates can explain the positive effect of detergents on the refolding yield. Both ionic and non—ionic detergents can be used to suppress aggregation upon dilution of the denatured protein in the renaturation buffer. Using laurylmaltosid, Chaps (3~[3- chloramidopropyl] dimethylammonia-l-propane sulfonate) or some other detergents during renaturation, the yield of refolded protein can be improved [32—34,P4]. Other 0 \/ u “T’fifiaiif D’ehatified'o—‘glttcosi'dase*fu SCd'tO“ a—poly—a 500 Expression vectors and delivery systems / detergents, inhibiting renaturation, have to be removed after dilution by addition of cyclodexrrin, which specifically binds detergent molecules [35]. Anorher possibility for suppressing unspecific intermolecu— lar interactions is the coupling of the denatured prOtein to a tag was bound to Heparin—Sepharose. Renaturation under conditions at which the protein is still bound to the matrix resulted in high yields of active protein even at protein con- centrations in a mg/ml range [36]. Another matrix used for this kind of renaturation was Ni-NTA agarose, originally developed for an efficient protein purification. After binding the denatured protein to the matrix via a His-tag the column is equilibrated with renaturation buffer and the refolded protein can be eluted by an imidazole or pH gradient. First demonstrated for chloramphenicol acetyl transferase, recently, the oxidative renaturation of a disulfide bridged prOtein TIMP 3 on a Ni-agarose was described [37,38]. Conclusions Todays detailed knowledge of the mechanisms of protein folding and its off—pathway reactions, aswell as the interre- lation of protein stIuCture and folding, makes it possible to basically design renaturation experiments. Still, the specif- ic conditions regarding buffer composition, protein concentration, temperature, and so on, has to be optimized for every protein. Failure of renaturation may be caused by omission of cofactors, such as structurally important metal ions, or degradation by traces of proteases. For those pro- teins which are synthesized as proforms in vivo, the prosequence may be crucial for structure formation and has to be included in the refolding scheme [39]. Strucmral and functional analyses of proteins, especially for therapeutic or other industrial applications, require large amounts of pro- teins. Inclusion body production and protein renaturation provides an efficient route to meet these requirements. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: . ' of special interest " of outstanding interest 1. Gribskov M, Burgess RR: Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene 1983, 26:109-1 18. 2. Kiefhaber T, Rudolph R. Kohler H-H. Buchner J:-Protein aggregation in vitro and in vivo: a quantitative model of the kinetic competition between folding and aggregation. Bio/Technology 1991, 9:825-829. 3. Schein CH. Noteborn MHM: Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/Technology 1988, 6:291 -294. 4. Hlavac F, Rouer E: Expression of the protein-tyrosine kinase p56l'ck by the pTRX vector yields a highly soluble protein recovered by mild sonication. Protein Expr Purif 1997, 11 :227-232. 5. Oswald T, Wende W, Pingoud A, Rinas U: Comparison of N-terminal affinity fusion domains: effect on expression level.and product - heterogeneity of recombinant restriction endonuclease EcoRV. Appl Microbiol Biotechnol 1994, 42:73-77. 6. Goloubinoff P, Gatenby AA, Lorimer GH: GroE heat shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. Nature 1989, 337:44-47. rginine_v_fi_.._...-.. _ g . 8. Cole PA: Chaperone-assisted protein expression. Structure 199’6j""""“""‘" “j WWW 7. Machida 8, Yu Y, Singh SP, Kim J-D, Hayashi K, Kawata Y: Overproduction of fi-glucosidase in active form. by an Escherichia coli system coexpressing the chaperonin GroEL/ES. FEMS Microbiol Lett 1998, 159:41-46. The expression of recombinant B-glucosidase with coexpression of the GroE system in E. coli was analysed. (in the absence of GroE, the recombinant protein was expressed in the insoluble fraction and constituted 80% of the total cellular protein. The coexpression of GroEL/ES leads to a significant fraction of soluble active B-glucosidase, which is even more increased at low induction temperatures. 11:239-242. 9. Thomas JG, Ayling A, Baneyx F: Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. Appl Biochem Biotechnol 1997, 66:197-238. 10. Yasukawa T, Kanei-lshii C, Maekawa T, Fujimoto J, Yamamoto T, lshii 8: Increase of solubility in Escherichia coli by coproduction of the bacterial thioredoxin. J Biol Chem 1995, 270:25328-25831. 11. Miele L, Cordella-Miele E, Mukherjee AB: High level bacterial expression of uteroglobin. a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure. J Biol Chem 1990, 265:6427-6435. 12. Georgiou G, Telford JN, Shuler ML, Wilson DB: Localization of inclusion bodies in Escherichia coli overproducing B-Iactamase or alkaline phosphatase. Appl Environ Microbiol1986. 5211571161. 13. Sone M, Akiyama Y. lto K: Differential in vivo roles played by DsbA - and Dst in the formation of protein disulfide bonds. J Biol Chem 1997, 272:1 0349-1 0352. Using a mutant form of alkaline phosphatase, disulfide bond formation in the periplasm of E. coli was investigated. It was observed that in wild-type cells an incorrect disulfide was formed first, which was subsequently converted to the native one. This conversion did not occur in Dst-disrupted cells. in DsbA-disrupted cells, the disulfide formation was less efficient, but the disulfides formed were predominantly native ones. 14. Maskos K: The bifunctional a-amylase/trypsin inhibitor from Ragi: 3-dimensional structure, inhibitory properties and oxidative folding in vivo and in vitro [PhD Thesis]. Dissertation no: 11399. Zurich: ETM;1995. 15. Wunderlich M, Glockshuber R: In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide- isomerase DsbA. J Biol Chem 1993, 268:24547-24550. 16. Oberg K, Chrunyk BA, Wetzel R, Fink AL: Native like secondary structure in interleukin-1 beta inclusion bodies by attenuated total reflectance FTIR. Biochemistry 1994, 33:2628-2634. 17. Zettlmeissl G, Rudolph R, Jaenicke R: Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. Biochemistry 1979,18:5567-5571. 18. Mukhopadhyay A: Inclusion bodies and purification of proteins in biologically active forms. Adv Biochem Eng Biotechnol 1997, 56161-1 09. 19. Rudolph R, Bbhm G, Lilie H, Jaenicke R: Folding proteins. in Protein - Function, a Practical Approach, edn 2. Edited by Creighton TE. Oxford: lRL Press; 1997:57-99. This chapter contains protocols for inclusion body preparation and renaturation of solubilized proteins. Methods for disulfide bond formation are discussed. General monitoring of protein folding and association reactions are considered. 20. Maachupalli-Reddy J, Kelley BD, De Bernardez Clark E: Effect of - inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme. Biotechnol Prog 1997, 13:144-150. Non-proteinaceous and protein contaminants were analyzed with respect to their efffect on the renaturation of lysozyme. Whereas DNA, lipopolysaccharides and phospholipids showed only marginal influence protein, contaminants can cause coaggregation of lysozymeI thus decreasing the yield of renaturation. A kinetic analysis of aggregation and ' folding is given. 21. Rudolph R: Renaturation of recombinant, disulfide-bonded proteins from inclusion bodies. In Modern Methods in Protein and Nucleic Acid Research. Edited by Tschesche H. New York: Walter de Gryter; 1990:149-172. 22. Ahmed AK, Schaffer SW, Wetlaufer DB: Nonenzymatic reactivation of reduced bovine pancreatic ribonuclease by air oxidation and by . _...W~._~_._w__eAna/.Biochemig92,.2052263:270- w 25. 27. 23. 24'. 2e. ' 28. 29. 30. Advances in refolding of proteins produced in E. coli Lilie, Schwarz and Rudolph 501 .l‘ glutathione oxidoreduction buffers. J Biol Chem 1975, 250:8477-8482. Wetlaufer DB. Branca PA, Chen GX: The oxidative folding of proteins by disulfide plus thiol does not correlate with redox potential. Protein Eng 1987, 2:141-146. BuchnerJ, Pastan I, Brinkmann U: A method for increasing the yield of properly folded recombinant fusion proteins: single-chain immunotoxins from renaturation of bacterial inclusion bodies. Moroder L, Besse D, Musiol HJ, Rudolph-Bohner S, Siedler F: Oxidative folding of cystine-rich peptides vs regioselective cysteine pairing strategies. Biopolymers 1996. 40:207-234. Hofmann A, Tai M, Wong W, Glabe CG: A sparse matrix screen to establish initial conditions for protein renaturation. Anal Biochem 1995, 23028-15. Rudolph R, Lilie H: In vitro folding of inclusion body proteins. FASEB J1996,10:49-56. Buchner J, Rudolph R: Renaturation, purification and characterization of recombinant Fab-fragments produced in Escherichia coli. Bio/Technology 1991, 9:157-162. Lin WJ, Traugh JA: Renaturation of casein kinase II from recombinant subunits produced in Escherichia coli: purification and characterization of the reconstituted holoenzyme. Protein Expr Purif1993, 41256-264. Hsih MH, Kuo JC, Tsai HJ: Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli. Appl Microbiol Biotechnol 1997, 48:66-72. Lin TY. Timasheff SN: On the role of surface tension in the stabilization of globular proteins. Protein Sci 1996, 5:372—381. . Wetlaufer DB, Xie Y: Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase 11. Protein Sci 1995, 4:1535-1543. 33. Tandon S, Horowitz PM: Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. J Biol Chem 1987, 262:4486-4491. 34. Goldberg ME, Expert-Bezancon N, Vuillard L, Rabilloud T: Ncn- " detergent sulfobetaines: a new class of molecules that facilitate protein renaturation. Fold Des 1996, 1:21-27. ' 35. Rozema D, Gellman SH: Artificial chaperone-assisted refolding of carbonic anhydrase B. J Biol Chem 1996, 271 :3478—3487. 3Ens—StempfeLG,.HollzNeugebauerfi,‘Rudolph R: lmproved_rgfolding of an immobilized fusion protein. Nat Biotechnol 1996, 14:329-334. ‘ 37. Holzinger A, Phillips KS. Weaver TE: Single-step purification/solubilization of recombinant proteins: application to surfactant protein B. Biotechniques 1996, 20:804-806. 38. Negro A, Onisto M, Grassato L, Caenazzo C. Garbisa S: Recombinant human TlMP-3 from Escherichia coli: synthesis. refolding. physico-chemical and functional insights. Protein Eng 199210593699. 39. Shinde U, Inouye M: Propeptide-mediated folding in subtilisin: the intramolecular chaperone concept. Adv Exp Med Biol 1996, 379:147-154. Patents P1. Glockshuber R. Wunderlich M, Skerra A, Rudolph R: Verbessereung der Ausbeute bei der Sekretion von disulfidverbrfickten Proteinen. European Patent 1992. 0 510 658 B1. P2. Fischer 8, Rudolph R. Mattes R: Process for the activation of gene- technologically produced, heterologous eukaryotic proteins after . expression in prokaryotes. European Patent 1986, 398 725 A1. P3. Builder 8, Ogez JR: Purification and activity assurance of precipitated heterologous proteins. US Patent 1986, 4 620 948. P4. Cerletti N, McMaster G, Cox D, Schmitz A, Meyhack B: Process for the production of biologically active protein. European Patent 1990, O 433 225 A1. ...
View Full Document

Page1 / 5

Refolding Proteins from E. Coli -...

This preview shows document pages 1 - 5. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online