EP1430078A2 - Procede pour augmenter la solubilite, le taux d'expression et l'activite de proteines pendant la production par recombinaison - Google Patents

Procede pour augmenter la solubilite, le taux d'expression et l'activite de proteines pendant la production par recombinaison

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Publication number
EP1430078A2
EP1430078A2 EP02774617A EP02774617A EP1430078A2 EP 1430078 A2 EP1430078 A2 EP 1430078A2 EP 02774617 A EP02774617 A EP 02774617A EP 02774617 A EP02774617 A EP 02774617A EP 1430078 A2 EP1430078 A2 EP 1430078A2
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EP
European Patent Office
Prior art keywords
dnak
lysate
proteins
helper proteins
helper
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02774617A
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German (de)
English (en)
Inventor
Manfred Watzele
Regina Schweizer
Cordula Nemetz
Robin Steigerwald
Thomas Emrich
Katrin Zaiss
Erhard Fernholz
Baerbel Walckhoff
Hans Joachim Schoenfeld
Birgit Offen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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Application filed by F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Publication of EP1430078A2 publication Critical patent/EP1430078A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the present invention relates to a method for producing a lysate containing helper proteins, wherein a strain which is suitable for the production of in vitro translation lysates is transformed with a vector containing one or more genes coding for one or more helper proteins, wherein the helper proteins are expressed in this strain and the lysate containing helper proteins is obtained from these strains.
  • the present invention furthermore relates to a lysate containing helper proteins obtainable by the process according to the invention, blends from these lysates and the use of the lysates and blends in in vitro translation systems.
  • helper proteins have already been described in the prior art.
  • the application WO 94/24303 describes the use of DnaJ, DnaK, GrpE, GroEL and GroES for activating an in vitro synthesized protein.
  • a cell-free extract is described which is largely free of protein and DNA-degrading enzymes and the incubation in an in vitro transcription / translation medium which contains helper proteins.
  • helper proteins are listed in the various cell-free extracts of common in vitro transcription / translation approaches from E. coli, rabbit reticulocytes and wheat germ.
  • helper proteins are therefore added, or the helper proteins present in the lysate are used.
  • the addition of purified helper proteins is uneconomical, while the helper proteins present in the lysates are generally not sufficient to adequately protect proteins from aggregation and misfolding.
  • EP 0885967 A2 describes the coexpression of DnaJ, Dnak and GrpE helper proteins in a cellular expression system to improve protein folding.
  • helper proteins are disadvantageous since the synthesis potential of the expression system must be distributed to other proteins in addition to the protein to be expressed.
  • RNA, 6, 778 describes that the human telomerase, consisting of the catalytic subunit hTERT and the RNA hTR involved, was produced in vitro with a rabbit reticulocyte system and in vivo in active form in yeast cells.
  • telomere expression in the cell-free rabbit reticulocyte system cannot be carried out on a large scale, since this requires large amounts of lysate, which are expensive to produce. Another reason that speaks against this is animal welfare. Masutomi et al. (2000), J. Biol. Chem., 275, 22568 describe the expression of hTERT in insect cells and the reconstitution with hTR transcribed in vitro. However, they point out that all methods of synthesizing telomerase in bacterial expression systems have failed.
  • the object was therefore to develop a process which makes it possible to provide helper proteins in an optimal and economical manner in the in vitro synthesis of a protein (hereinafter also referred to as target protein).
  • the addition of the helper proteins is to be optimized in such a way that the protein (target protein) synthesized in vitro is adequately protected against aggregation and misfolding.
  • the present object was achieved by a method for producing a lysate containing helper proteins, characterized in that
  • a strain which is suitable for obtaining in vitro translation lysates is transformed with a vector containing one or more genes coding for one or more helper proteins, that the helper proteins are expressed in this strain and that the lysate containing helper proteins from them Tribes is won.
  • This lysate according to the invention is then present during the in vitro synthesis of the target protein.
  • Helper proteins in the sense of the invention are proteins which increase the solubility, the folding and / or the activity of the proteins expressed in vitro and, in some cases, can also increase their expression rate.
  • a soluble protein in the sense of the invention means that the protein from the reaction mixture remains in the supernatant after a 2-minute centrifugation at 10,000 times the acceleration of gravity g and does not sediment.
  • An increase in the solubility in the sense of the invention means that when the helper proteins are added, a higher proportion of the protein (at least 10%) remains in solution than when the helper proteins are not added.
  • helper proteins are so-called heat shock proteins and chaperones such as those from the DnaK or GroE system, chaperonins, protein disulfide isomerase, trigger factor and prolyl-cis-trans isomerase.
  • the folding helper proteins are selected from one or more of the following protein classes: Hsp60, Hsp70, Hsp90, HsplOO protein family, family of small heat shock proteins and isomerases.
  • Molecular chaperones represent the largest group of folding-assisting proteins and are understood according to the invention as folding helper proteins (Gething and Sambrook, 1992; Hartl, 1996; Buchner, 1996; Bschreibinger and Buchner, 1998). Because of their overexpression under stress conditions, most molecular chaperones can also be classified in the group of heat shock proteins (Georgopoulos and Welch, 1993; Buchner, 1996), this group is also understood according to the invention as a folding aid protein.
  • the group of molecular chaperones can be divided into five unrelated protein classes, the Hsp60, Hsp70, Hs ⁇ 90, HsplOO protein families and the family of small heat shock proteins, based on sequence homologies and the molecular masses (Gething and Sambrook, 1992; Hendrick and Hartl , 1993).
  • the best investigated chaperone at all is GroEL, a member of the Hsp ⁇ O family from E. coli.
  • the representatives of the Hsp60 family are also known as chaperonins and divided into two groups.
  • GroEL and its co-chaperone GroES and their strongly homologous relatives from other bacteria as well as mitochondria and chloroplasts form the group of I chaperones (Sigler et al., 1998; Fenton and Horwich, 1997).
  • the Hsp60 proteins from the eukaryotic cytosol and from archebacteria are grouped together as Group II chaperones (Gutsche et al., 1999).
  • the Hsp60 proteins show a similar oligomeric structure in both groups.
  • the co-chaperone GroES also forms a heptameric ring and binds in this form to the poles of the GroEL cylinder.
  • This binding of GroES leads to a limitation of the substrate binding depending on its size 10-55kDa; Ewalt et al., 1997).
  • the substrate binding is regulated by ATP binding and hydrolysis.
  • the Hsp70 proteins In addition to the representatives of the Hsp60 family, the Hsp70 proteins also bind to the nascent polypeptide chain (Beckman et al., 1990; Welch et al., 1997). There are usually several constitutively expressed and stress-induced representatives of the Hsp70 family in both prokaryotic and eukaryotic cells (Vickery et al., 1997; Kawula and Lelivelt, 1994; Fink, 1997; Welch et al., 1997). In addition to protein folding directly on the ribosome, these are also involved in the translocation of proteins across cell and organelle membranes (Schatz & Doberstein, 1996).
  • Hsp70 co-chaperones
  • Hsp90 is one of the most expressed proteins (Welch and Feramisco, 1982).
  • the representatives of this family act primarily in multimeric complexes, recognizing a large number of important signal transduction proteins with native-like structures. By binding to Hsp90 and its partner proteins, these structures are stabilized and thus the binding of ligands to the signal proteins is facilitated. In this way, the substrates can achieve their active conformation (Sullivan et al., 1997; Bohen et al., 1995; Buchner, 1999).
  • HsplOO chaperones in particular have been distinguished by their ability to dissolve aggregates that have already formed in cooperation with the Hsp70 chaperones (Parsell et al., 1994; Golloubinoff et al., 1999; Mogk et al., 1999). While their main job is the Mediation of thermotolerance appears to be (Schirmer et al, 1994; Kruger et al., 1994), some representatives such as ClpA and ClpB together with the protease subunit ClpP mediate the proteolytic degradation of proteins (Gottesman et al., 1997).
  • the fifth class of chaperones the small heat shock proteins (sHsps) represent a very divergent family of heat shock proteins that are found in almost all organisms.
  • the name for this family of chaperones is their relatively low monomeric molecular weight of 15-40 kDa.
  • sHsps mostly exist as highly oligomeric complexes with up to 50 subunits, from which molecular masses of 125 kDa up to 2 MDa have been observed (Spectof et al., 1971; Arrigo et al., 1988; Andreasi-Bassi et al., 1995; Ehrnsperger et al., 1997).
  • sHsps can suppress the aggregation of proteins in vitro (Horwitz, 1992; Jakob et al., 1993; Merck et al, 1993; Jakob and Buchner, 1994, Lee et al., 1995; Ehrnsperger et al., 1997b ).
  • SHsps bind up to one substrate molecule per subunit and are therefore more efficient than the model chaperone GroEL (Jaenicke and Creighton, 1993; Ganea and Harding, 1995; Lee et al., 1997; Ehrnsperger et al, 1998a). Under stress conditions, the binding of non-native protein to sHsps prevents the irreversible aggregation of the proteins.
  • the proteins are kept in a soluble folding-competent state by binding to sHsps. After the restoration of physiological conditions, the non-native protein of ATP-dependent chaperones such as Hsp70 can be released from the complex with sHsp and reactivated.
  • the isomerases used for the process according to the invention are, for example, folding catalysts from the class of the peptidyl-prolyl-cis / trans isomerases and representatives of the disulfide isomerases.
  • Folding helper proteins that function in the same or similar manner as the folding helper proteins described above are also encompassed by the present invention.
  • the method according to the invention is particularly preferred if the strain has been transformed with different vectors, the vectors differing at least in that the genes contained therein code for different helper proteins.
  • the vectors differing at least in that the genes contained therein code for different helper proteins.
  • the strain which is suitable for obtaining in vitro translation lysates additionally has at least one of the following properties: poor or deficient in RNAse, poor in or exonuclease, deficient, poor in protease or deficient cient.
  • An embodiment of the invention includes the method according to the invention, wherein the lysate is obtained in such a way that, in addition to the helper proteins, all components are contained in the lysate which are necessary for in vitro translation or for in vitro transcription / translation of a target protein. At least the following components are required for in vitro translation or for in vitro transcription / translation of a target protein:
  • Such primary energy donors are, for example, acetyl phosphate, creatine phosphate, phosphoenolpyruvate, pyruvate, glucose or other possible substrates known to the person skilled in the art, which are reacted directly or via several enzyme-catalyzed intermediate steps, so that molecules with an energy-rich phosphate bond are formed, which then transfer this phosphate group onto a nucleotide monophosphate or can transfer a nucleotide diphosphate.
  • the present invention thus also relates to a lysate containing helper proteins, this lysate being obtainable by the process according to the invention.
  • the lysate according to the invention can be obtained, e.g. B. Methods in which the promoters of the helper protein genes naturally occurring in the strains are changed so that a larger amount of the helper protein is formed.
  • Another method is the transformation of a stem with a piece of DNA, which the co- dated helper protein and which is integrated into the genome of the strain one or more times, to then be amplified by this strain during cell division. Any lysate that has the same properties as the lysate obtainable by the method according to the invention is encompassed by the present invention.
  • the lysate described above is preferred, at least two different helper proteins being contained,
  • the invention also includes lysates essentially containing a helper protein.
  • helper proteins being selected from the following group:
  • Blends from various lysates according to the invention can prove to be very particularly advantageous.
  • the number of helper proteins and their concentration could be optimized for the respective in vitro translation or in vitro transcription and translation of the target protein.
  • a preferred embodiment is blending from one or more lysates according to the invention with a lysate containing all components which are required for in vitro translation or for in vitro transcription / translation.
  • the invention furthermore relates to a strain which is suitable for obtaining in vitro translation lysates and which has been transformed with a vector comprising one or more genes coding for one or more helper proteins.
  • the present invention also relates to the use of a lysate according to the invention or a blend according to the invention for in vitro translation or for in vitro Transcription / translation.
  • the invention further comprises the use of a lysate according to the invention or a blend according to the invention in a CECF or CFCF reactor.
  • Such an experimental arrangement is realized in the principles of "continuous exchange cell-free” (CECF) or “continuous flow cell-free” (CFCF) protein synthesis (US 5,478,730; EPA 0 593 757; EPA 0 312 612; Baranov & Spirin (1993) Meth. Enzym. 217, 123-142).
  • CECF reactors consist of at least two discrete chambers, which are separated from each other by a porous membrane. The high molecular weight components are retained in the reaction chamber by this porous interface, while low molecular weight components are exchanged between the reaction chamber and the supply chamber.
  • CFCF processes a supply solution is pumped directly into the reaction chamber and the final reaction products are pressed out of the reaction compartment through one or more ultrafiltration membranes.
  • Corresponding reactor principles have been implemented in "continuous exchange cell-free” (CECF) and “continuous flow cell-free” (CFCF) protein synthesis (US 5,478,730; EPA 0 593 757; EPA 0 312 612; Baranov & Spirin (1993 ) Meth. Enzyme. 217, 123-142).
  • the expression of the target proteins could also be increased significantly by adding the helper proteins.
  • the target proteins can be all types of pro- and eukaryotic proteins and also archaeal proteins.
  • the problem with in vitro transcription / translation systems has been, in particular, the expression of secretory proteins and membrane proteins, especially when there is no sufficient amount of folding helper proteins.
  • the successful expression of lipoproteins and membrane-bound proteins has been described in the prior art, but was subject to significant limitations (Hupa and Ploegh, 1997; Falk et al., 1997).
  • the method according to the invention can be suitable in particular for the expression of lipoproteins and membrane-bound proteins or secretory proteins as the target protein, since folding assistant proteins are made available in sufficient quantity by the coexpression.
  • telomere could be produced by adding the lysates according to the invention to a cell-free in vivo translation system. So far, active telomerase has not been expressed in prokaryotic cells or in cell-free prokaryotic lysates.
  • the present invention thus also relates to the use of a lysate according to the invention or a blend according to the invention for in vitro Translation or for in vitro transcription / translation of telomerase.
  • the cell-free in vitro expression of telomerase with prokaryotic lysates could be achieved according to the invention by adding a lysate according to the invention containing the helper proteins DnaK and DnaJ to a conventionally produced E.
  • telomere Since the telomerase is expressed in all eukaryotes from yeast to human, the analysis of the "pure" telomerase is difficult since, in principle, there are always additional cofactors from the expression systems.
  • Soluble telomerase in E. coli lysates from two different preparations in liquid or lyophilized state with and without the addition of helper proteins.
  • Soluble telomerase content in lysates from the non-transformed A19 strain which were blended with 25% or 50% lysate from the A19 strain which had been transformed with a plasmid coding for the proteins from the DnaK system.
  • Lane 1 and 3 each show the supernatant fractions, lane 2 and 4 the precipitation fractions.
  • Figure 11
  • pIVEX2.3-GFP The gene for the green fluorescent protein from Aequoria victoria (Prasher et al. (1992) Gene 111, 229) was cloned into the ⁇ IVEX2.3 vector (Röche Diagnostics GmbH Mannheim, Germany) via the Ncol interface.
  • pIVEX2.4b-Mal-Epo The gene for the maltose-binding protein was isolated from pMAL-p2 (New England Biolabs, Beverley, MA, USA) and won into the vector pIVEX2.4b. The gene for human erythropoietin (Jacobs et al. (1985) Nature 313, 806) was mapped behind this gene without the signal sequence, giving rise to pIVEX2.4b-Mal-Epo.
  • telomere vector The gene for the catalytic subunit of human telomerase (Autexier C. et al. (1996) EMBO Journal. 15, 5928) was cloned into the ⁇ IVEX2.4bNde vector via the Nde 1 interface, where ⁇ IVEX2.4bNde -hTERT was created.
  • NEX2.4-Rhodanese The bovine mitochondrial Rhodanese gene (Miller DM et al. (1991), J. Biol Chem. 266, 4686) was cloned into the pIVEX2.4 vector via the Nco I interface, where pIVEX2. 4-Rhodanese was born.
  • DRDKIG which codes for the proteins Dna-K, Dna-J and GrpE (Dale GE et al. (1 94) Protein Eng. 7, 925), as well as purified Dna-K, Dna-J and GrpE protein was developed by Dr. H. Schönfeld Hoffmann-La Röche Ltd., Basel Switzerland.
  • pREP4-groESL which was coded for the Gro-EL and Gro-ES proteins, was obtained from P. Caspers (Caspers et al. (1994) Cell Mol. Biol. 40, 635-44). Purified GroEL and GroES protein was developed by Dr. H. Schönfeld Hoffmann-La Röche Ltd., Basel Switzerland.
  • the lysate was prepared with an E. coli A19 strain using the Zubay method (Annu. Rev. Genet. (1973) 7, 267).
  • Example la Influence of helper proteins on the solubility of telomerase
  • the prVEX2.4bNde-hTERT plasmid was used in the bacterial in vitro expression system with and without the addition of 1 ⁇ M of the helper proteins DnaK, DnaJ and GrpE.
  • the Rapid Translation System RTS 500 E. coli circular template kit (Röche Diagnostics GmbH) was used for the expression.
  • the helper proteins were added in a purified form.
  • the reaction products were then centrifuged at 100,000 g for 2 min.
  • the resulting liquid and the supernatant were taken up in SDS sample buffer and applied to an SDS gel.
  • the SDS gel was analyzed by Western blot. The amounts of protein detected can be seen in FIG. 1.
  • Example 1b Influence of helper proteins of the DnaK system on the solubility of a fusion protein consisting of maltose binding protein and erythropoietin
  • the fusion protein was synthesized by the expression vector pIVEX2.4b-Mal-Epo in the bacterial in vitro expression system with and without the addition of 1 ⁇ M each of the helper proteins DnaK, GrpE and DnaJ (analogously to example la).
  • the reaction products were then centrifuged at 100,000 g for 2 min.
  • the resulting PeUet and the supernatant were taken up in SDS sample buffer and applied to an SDS gel.
  • Example 2 Influence of helper proteins in different lysate preparations and lysate lyophilization
  • E. coli lysates from 2 different preparations were in a liquid or lyophilized state with and without the addition of 1 ⁇ M of the helper proteins DnaK, DnaJ and GrpE in one Telomerase expression used.
  • Example 4 A mixture of DnaK and DnaJ is sufficient
  • Example 5 Influence of helper proteins of the DnaK system on the activity of the green fluorescent protein (GFP)
  • Wüdtyp GFP was expressed without the oxygen required for folding analogously to Example 4 by filling the reaction vessel from the RTS 500 kit up to the top. After the reaction had ended, the reaction product was pipetted into an open vessel and in the presence of air-oxygen for 24 Stored in the refrigerator for hours. During this time, the correctly folded portion of the GFP protein can oxidize and thus emit the fluorophore. The activity of the GFP protein was then measured from the fluorescence.
  • Example 6 Increasing the amount of synthesis of telomerase depending on the addition of helper proteins
  • Example 4 Analogously to Example 4, amounts of 1 ⁇ M, 2 ⁇ M and 3 ⁇ M of the two helper proteins DnaK and DnaJ were used in the telomerase expression. However, the reaction products were now centrifuged at 100,000 g for 30 min and the fractions were analyzed in a Western blot.
  • the total amount of telomerase synthesized increased significantly in the case of outside preparations with helper protein. In the approach with 3 ⁇ M DnaK / DnaJ, the increase was even more than 50% (FIG. 7).
  • Example 7 Measurement of the activity of reconstituted telomerase as a function of helper proteins.
  • telomerase was expressed in the presence of 0 ⁇ M, 2 ⁇ M and 10 ⁇ M each on DnaK and DnaJ. The batches were then reconstituted with the RNA component and an activity test was carried out using the Telo TAGGGG Telomerase PCR ELISA (Röche Diagnostics GmbH). The telomerase was reconstituted according to the procedure of Weinrich S.L. et al. (1997) Nature Genet. 17, 498.
  • helper proteins were first heat-treated at 70 ° C. for 30 min before being used in the in vitro protein synthesis.
  • Table 1 the helper proteins were first heat-treated at 70 ° C. for 30 min before being used in the in vitro protein synthesis.
  • helper proteins With helper proteins, the activity could be increased more than tenfold compared to the approach without helper proteins.
  • the heat-treated helper proteins were completely inactive.
  • Example 8 Preparation of helper protein producing strains.
  • strain A19 and the strain Xl-Blue were transformed with plasmids (see under A.) which either contain the helper proteins from the DnaK / DnaJ / GrpE system or from the GroEL / ES system behind an IPTG inducible promoter.
  • Strain A19 has a mutation with RNase I gene, while strain Xl-Blue has a deficiency in protease genes.
  • the transformed cells were grown on LB medium and induced at an optical density of 1.0 measured at 600 nm wavelength for 30 min with IPTG (final concentration up to 1 mM).
  • Example 9a Use of lysates from the cells transformed with the DnaK / DnaJ / GrpE system
  • telomere lysates from the cells transformed with the DnaK / DnaJ / GrpE system were then used for in vitro translation with the telomerase gene.
  • the lysates from the untransformed strains were used. It could be shown that, in contrast to the untransformed strains, 100% soluble telomerase could be expressed with the lysate from the IPTG-induced transformed strains (FIG. 8).
  • Example 9c Use of lysates prepared from the cells which were transformed with pREP4-groESL
  • bovine Rhodanese was expressed using the pINEX2.4-Rhodanese plasmid (24 h, 30 ° C), the expression once without Addition of transformed lysate (conditions as specified in the manufacturer's product description), the other time with addition of 50% of a lysate (from cells which had been transformed with pREP4-groESL plasmid and had overexpressed GroEL and GroES by incubation) , The reaction mixtures were then centrifuged for 5 min at 10,000 ⁇ g, the resulting pellet and the supernatant were taken up in SDS sample buffer, separated on an SDS gel and stained with Coomassie Blue (see FIG. 10).
  • Rhodanese also increases significantly with the addition of the lysate containing helper protein (GroEL / ES) (see FIG. 11).

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Abstract

La présente invention concerne un procédé de production d'un lysat contenant des protéines auxiliaires. Selon ledit procédé, on transforme une souche, qui convient à l'obtention de lysats de traduction <i>in vitro</i>, avec un vecteur contenant au moins un gène codant pour au moins une protéine auxiliaire, les protéines auxiliaires étant exprimées dans cette souche et le lysat contenant les protéines auxiliaires étant extrait de ces souches. L'invention concerne également un lysat contenant des protéines auxiliaires pouvant être obtenu selon le procédé présenté, des mélanges de ces lysats et l'utilisation des lysats et de leurs mélanges dans des systèmes de traduction <i>in vitro</i>.
EP02774617A 2001-09-17 2002-09-14 Procede pour augmenter la solubilite, le taux d'expression et l'activite de proteines pendant la production par recombinaison Withdrawn EP1430078A2 (fr)

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DE10145694A DE10145694A1 (de) 2001-09-17 2001-09-17 Verfahren zur Erhöhung der Löslichkeit, der Expressionsrate und der Aktivität von Proteinen während der rekombinanten Herstellung
DE10145694 2001-09-17
PCT/EP2002/010329 WO2003025116A2 (fr) 2001-09-17 2002-09-14 Procede pour augmenter la solubilite, le taux d'expression et l'activite de proteines pendant la production par recombinaison

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US20040248238A1 (en) 2004-12-09
WO2003025116A2 (fr) 2003-03-27
WO2003025116A3 (fr) 2004-03-11
JP2005503157A (ja) 2005-02-03

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