EP1430078A2 - Method for increasing the solubility, expression rate and the activity of proteins during recombinant production - Google Patents

Method for increasing the solubility, expression rate and the activity of proteins during recombinant production

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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)
French (fr)
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/en
Withdrawn legal-status Critical Current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

The invention relates to a method for producing a lysate containing auxiliary proteins. According to said method, a strain, which is suitable for obtaining in vitro translation lysates is transformed using a vector containing one or more genes that code for one or more auxiliary proteins, the auxiliary proteins being expressed in said strain and the lysate containing auxiliary proteins being obtained from said strains. The invention also relates to a lysate containing auxiliary proteins that can be obtained according to the inventive method, to blends of said lysates and to the use of the lysates and blends in in vitro translation systems.

Description

Verfahren zur Erhöhung der Löslichkeit, der Expressionsrate und der Aktivität von Proteinen während der rekombinanten HerstellungProcess for increasing the solubility, expression rate and activity of proteins during recombinant production
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung eines Lysates enthaltend Helferproteine, wobei ein Stamm, welcher geeignet ist für die Gewinnung von in-vitro Translations-Lysaten, transformiert wird mit einem Vektor enthaltend ein oder mehrere für ein oder mehrere Helferproteine kodierende Gene, wobei die Helferproteine in diesem Stamm ex- primiert werden und wobei das Lysat enthaltend Helferproteine aus diesen Stämmen gewonnen wird. Des weiteren ist Gegenstand der vorliegenden Erfindung ein Lysat enthaltend Helferproteine erhältlich nach dem erfindungsgemäßen Verfahren, Verschnitte aus diesen Lysaten und die Verwendung der Lysate und Verschnitte in in vitro Translationssystemen.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.
Die Zugabe von hoch gereinigten Helferproteinen wurde im Stand der Technik bereits beschrieben. So ist in der Anmeldung WO 94/24303 die Verwendung von DnaJ, DnaK, GrpE, GroEL und GroES zur Aktivierung eines in vitro synthetisierten Proteins beschrieben. Beschrieben wird ein Zeil- freier Extrakt, der weitgehend frei von Protein- und DNA-abbauenden Enzymen ist und die Inkubation in einem in vitro Transkriptions/Translations-Medium, welches Helferproteine enthält.The addition of highly purified helper proteins has 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.
In WO 94/24303, wie auch in Kudlicki et al. (1995), J. Bacteriol. 177, 5517 und Kudlicki et al. (1996), J. Biol. Chem. 271, 31160 wird ein isolierter Komplex aus Ribosomen und einem Pep- tid/Protein durch Zugabe von Helferproteinen und ATP wieder aufgelöst, wobei das aktive Protein freigesetzt wird.In WO 94/24303, as also in Kudlicki et al. (1995), J. Bacteriol. 177, 5517 and Kudlicki et al. (1996), J. Biol. Chem. 271, 31160, an isolated complex of ribosomes and a peptide / protein is redissolved by adding helper proteins and ATP, the active protein being released.
Ryabova et al. (1997), Nature Biotechnology 15, 79 beschreiben den Einsatz von DnaJ, DnaK, GrpE, GroEL und GroES im Zusammenspiel mit Proteindisulfidisomerase bei der in vitro Translation mit einem E. coli Lysat. Die Zugabe von DnaJ, DnaK, GrpE allein führte zur Erhöhung der Löslichkeit eines Disulfid-haltigen Proteins, während die Zugabe von Proteindisulfidisomerase zu einer Erhöhung der Aktivität führte.Ryabova et al. (1997), Nature Biotechnology 15, 79 describe the use of DnaJ, DnaK, GrpE, GroEL and GroES in interaction with protein disulfide isomerase in in vitro translation with an E. coli lysate. The addition of DnaJ, DnaK, GrpE alone increased the solubility of a disulfide-containing protein, while the addition of protein disulfide isomerase led to an increase in activity.
Merk et al. (1999), J. Biochem. 125, 328 beschreiben den Einsatz von DnaJ, DnaK, GrpE, GroEL und GroES im Zusammenspiel mit Proteindisulfidisomerase bei der gekoppelten oder verknüpf- ten in vitro Transkription/Translation mit einer E. coli Ribosomen Fraktion. Die Zugabe der Helferproteine führte zu einer erhöhten Löslichkeit und zu einer erhöhten Aktivität der Proteine.Merk et al. (1999), J. Biochem. 125, 328 describe the use of DnaJ, DnaK, GrpE, GroEL and GroES in interaction with protein disulfide isomerase in the coupled or linked ten in vitro transcription / translation with an E. coli ribosome fraction. The addition of the helper proteins led to increased solubility and increased activity of the proteins.
In Fedorof & Baldwin (1998) Meth. Enzymol.290, 1 sind Helferproteine in den verschiedenen Zeil-freien Extrakten gebräuchlicher in vitro Transkriptions/Translations- Ansätze aus E. coli, Kaninchen-Retikulozyten und Weizenkeim aufgeführt.In Fedorof & Baldwin (1998) Meth. Enzymol. 290, 1 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.
In Fedorof & Baldwin (1997) J. Biol. Chem. 272, 5 wird die Bedeutung verschiedener Helferproteine bei der Kotranslationalen Proteinfaltung und die (oben erwähnten) Beispiele in der in vitro Proteinsynthese zusammengefaßt.Fedorof & Baldwin (1997) J. Biol. Chem. 272, 5 summarize the importance of various helper proteins in cotranslational protein folding and the (above-mentioned) examples in in vitro protein synthesis.
Im Stand der Technik werden also hochgereinigte Helferproteine zugesetzt, bzw. werden die in dem Lysat vorhandenen Helferproteine eingesetzt. Die Zugabe gereinigter Helferproteine ist unwirtschaftlich, während die in den Lysaten vorhandenen Helferproteine im allgemeinen nicht ausreichen, um Proteine ausreichend vor Aggregation und Mißfaltung zu schützen.In the prior art, highly purified 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 beschreibt die Koexpression von DnaJ-, Dnak-, GrpE-Helferproteinen in einem zellulären Expressions-System zur Verbesserung der Proteinfaltung.EP 0885967 A2 describes the coexpression of DnaJ, Dnak and GrpE helper proteins in a cellular expression system to improve protein folding.
Die Koexpression von Helferproteinen ist jedoch nachteilig, da das Synthesepotential des Expressionssystems neben dem zu exprimierenden Protein auf weitere Proteine verteilt werden muß.However, the co-expression of helper proteins is disadvantageous since the synthesis potential of the expression system must be distributed to other proteins in addition to the protein to be expressed.
In Bachand et al. (2000) RNA, 6, 778 wird beschrieben, daß die humane Telomerase, bestehend aus der katalytischen Untereinheit hTERT und aus der beteiligten RNA hTR, in vitro mit einem Kaninchen Retikulozyten System und in vivo in Hefezellen in aktiver Form hergestellt wurde.In Bachand et al. (2000) 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.
Holt et al. (1999) Genes & Development 13, 817 beschreiben, daß zur Rekonstitution von in vitro (Kaninchen Retikulozyten System) synthetisierter hTERT mit der beteiligten RNA hTR weitere Proteinfaktoren hsp 90 und p23 aus dem Retikulozytenextrakt als Helferproteine nötig sind.Holt et al. (1999) Genes & Development 13, 817 describe that for the reconstitution of hTERT synthesized in vitro (rabbit reticulocyte system) with the RNA hTR involved, further protein factors hsp 90 and p23 from the reticulocyte extract are necessary as helper proteins.
Die Expression von Telomerase im zellfreien Kaninchen Retikulozyten System ist für den Großmaßstab jedoch nicht durchführbar, da hierbei große Lysatmengen benötigt werden, welche teuer in der Herstellung sind. Ein weiterer Grund, der dagegen spricht, ist der Tierschutz. Masutomi et al. (2000), J. Biol. Chem., 275, 22568 beschreiben die Expression von hTERT in Insektenzellen und die Rekonstitution mit in vitro transkribierter hTR. Sie weisen jedoch darauf hin, daß alle Methoden, Telomerase in bakteriellen Expressionssystemen zu synthetisieren, gescheitert sind.However, the expression of telomerase 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.
Weinrich et al. (1997) Nat. Genet. 17, 498 erwähnen die erfolgreiche Synthese funktioneil aktiver Telomerase in einem Weizenkeim Transkriptions-Translationssystem. Das Weizenkeim Expressionssystem ist jedoch Erfahrungen wenig produktiv und liefert wegen dem Vorhandensein hoher Nuklease- und Proteaseaktivitäten größtenteils unvollständige Translationsprodukte.Weinrich et al. (1997) Nat. Genet. 17, 498 mention the successful synthesis of functionally active telomerase in a wheat germ transcription translation system. Experience has shown that the wheat germ expression system is not very productive and, because of the presence of high nuclease and protease activities, provides largely incomplete translation products.
Es bestand somit die Aufgabe, ein Verfahren zu entwickeln, daß es ermöglicht, bei der in vitro Synthese eines Proteins (im folgenden auch Zielprotein genannt) Helferproteine in optimaler und wirtschaftlicher Weise zur Verfugung zu stellen. Insbesondere soll die Zugabe der Helferproteine in der Weise optimiert werden, daß das in vitro synthetisierte Protein (Zielprotein) ausreichend vor Aggregation und Mißfaltung geschützt ist.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). In particular, 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.
Die vorliegende Aufgabe wurde gelöst durch ein Verfahren zur Herstellung eines Lysates enthaltend Helferproteine, dadurch gekennzeichnet,The present object was achieved by a method for producing a lysate containing helper proteins, characterized in that
daß ein Stamm, welcher geeignet ist für die Gewinnung von in vitro Translations-Lysaten, transformiert wird mit einem Vektor enthaltend ein oder mehrere für ein oder mehrere Helferproteine kodierende Gene, daß die Helferproteine in diesem Stamm exprimiert werden und daß das Lysat enthaltend Helferproteine aus diesen Stämmen gewonnen wird.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.
Dieses erfindungsgemäße Lysat ist dann während der in vitro Synthese des Zielproteins anwesend.This lysate according to the invention is then present during the in vitro synthesis of the target protein.
Helferproteine im Sinne der Erfindung sind dabei Proteine, welche die Löslichkeit, die Faltung und/oder die Aktivität der in vitro exprimierten Proteine erhöhen und dabei fallweise auch deren Expressionsrate steigern können. Ein lösliches Protein im Sinne der Erfindung bedeutet, daß das Protein aus dem Reaktionsansatz nach einer 2-minütigen Zentrifugation bei 10000-facher Erdbeschleunigung g im Überstand bleibt und nicht sedimentiert. Eine Steigerung der Löslichkeit im Sinne der Erfindung bedeutet, daß bei Zusatz der Helferproteine ein höherer Anteil des Proteins (mindestens 10%) in Lösung bleibt, als beim Ansatz ohne Zusatz der Helferproteine. Beispiele für Helferproteine sind sogenannte heat shock Proteine und Chaperone wie die aus dem DnaK oder GroE System, Chaperonine, Proteindisulfidisomerase, Trigger-Faktor und Prolyl-cis-trans Isomerase.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. Examples for 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.
Die Faltungshelferproteine sind aus einer oder mehreren der folgenden Proteinklassen ausgewählt: Hsp60-, Hsp70-, Hsp90-, HsplOO-Proteinfamilie, Familie der kleinen Hitzeschockproteine und Isomerasen.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.
Molekulare Chaperone stellen die größte Gruppe von faltungsassistierenden Proteine dar und werden erfindungsgemäß als Faltungshelferprotein verstanden (Gething und Sambrook, 1992; Hartl, 1996; Buchner, 1996; Beißinger und Buchner, 1998). Aufgrund ihrer Überexpression unter Stressbedingungen können die meisten molekularen Chaperone auch in die Gruppe der Hitzeschockproteine einordnet werden (Georgopoulos und Welch, 1993; Buchner, 1996), diese Gruppe wird erfindungsgemäß ebenfalls als Faltungshelferprotein verstanden.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; Beißinger 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.
Nachfolgend werden wichtige Faltungshelferproteine, die von der vorliegenden Erfindung umfaßt werden, näher erläutert. Die Gruppe der molekularen Chaperone läßt sich anhand von Sequenzhomologien und der molekularen Massen in fünf nicht verwandte Proteinklassen, die Hsp60-, Hsp70-, Hsρ90-, HsplOO-Proteinfamilien und die Familie der kleinen Hitzeschockproteine unterteilen (Gething und Sambrook, 1992; Hendrick und Hartl, 1993).Important folding helper proteins encompassed by the present invention are explained in more detail below. The group of molecular chaperones can be divided into five unrelated protein classes, the Hsp60, Hsp70, Hsρ90, 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).
• Hsp60• Hsp60
Das am Besten untersuchte Chaperon überhaupt ist GroEL, ein Vertreter der HspδO Familie aus E. coli. Die Vertreter der Hsp60 Familie werden auch als Chaperonine bezeichnet und in zwei Gruppen unterteilt. GroEL und sein Ko-Chaperon GroES und deren stark homologe Verwandte aus anderen Bakterien sowie Mitochondrien und Chloroplasten bilden die Gruppe der I Chaperone (Sigler et al., 1998; Fenton und Horwich, 1997). Die Hsp60 Proteine aus dem eukaryoti- schen Cytosol und aus Archebakterien werden als Gruppe II Chaperone zusammengefaßt (Gut- sche et al., 1999). In beiden Gruppen zeigen die Hsp60 Proteine einen ähnlichen oligomeren Aufbau. Im Falle von GroEL und den anderen Gruppe I Chaperoninen lagern sich 14 GroEL Untereinheiten zu einem Zylinder aus zwei heptameren Ringen zusammen, während die heptamere Ringstruktur bei den Chaperoninen der Gruppe II aus Archebakterien meist aus zwei unterschiedlichen Untereinheiten aufgebaut ist. Vertreter der Gruppe II Chaperonine aus dem eu- karyotischen Cytosol, wie z. B. der CCT-Komplex aus Hefe, sind hingegen aus 8 verschiedenen Untereinheiten mit genau definierter Organisation aufgebaut (Liou und Willison, 1997). Im zentralen Hohlraum dieses Zylinders können nicht native Proteine eingelagert und gebunden wer- den. Das Ko-Chaperon GroES bildet ebenfalls einen heptameren Ring und bindet in dieser Form an den Polen des GroEL- Zylinders. Diese Bindung von GroES führt allerdings zu einer Limitierung der Substratbindung in Abhängigkeit ihrer Größe 10-55kDa; Ewalt et al., 1997). Die Substratbindung wird dabei durch ATP-Bindung und Hydrolyse reguliert.The best investigated chaperone at all is GroEL, a member of the HspδO 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. In the case of GroEL and the other Group I chaperonins, 14 GroEL subunits are combined to form a cylinder made up of two heptameric rings, while the heptameric ring structure in group II chaperonins is usually made up of two different subunits from archebacteria. Representatives of group II chaperonins from the eukaryotic cytosol, such as. B. the yeast CCT complex, on the other hand, are made up of 8 different subunits with precisely defined organization (Liou and Willison, 1997). Non-native proteins can be embedded and bound in the central cavity of this cylinder the. 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.
• Hsp70• Hsp70
Neben den Vertretern der Hsp60 Familie binden auch die Hsp70 Proteine an die naszierende Polypeptidkette (Beckman et al., 1990; Welch et al., 1997). Sowohl in prokaryotischen als auch in eukaryotischen Zellen existieren meist mehrere konstitutiv exprimierte und stressinduzierte Vertreter der Hsp70 Familie (Vickery et al., 1997; Kawula und Lelivelt, 1994; Fink, 1997; Welch et al., 1997). Diese sind neben der Proteinfaltung direkt am Ribosom auch an der Translokation von Proteinen über Zeil- und Organellmembranen beteiligt (Schatz & Doberstein, 1996). Es wurde gezeigt, daß Proteine nur im ungefalteten bzw. teilgefalteten Zustand durch Membranen geschleust werden können (Hannavy et al., 1993). Während des Translokationsprozesses in Organellen sind vor allem Vertreter der Hsp70 Familie an der Entfaltung und Stabilisierung auf der cytosolischen Seite, als auch an der Rückfaltung auf der Organellseite beteiligt (Hauke und Schatz, 1997). Dabei ist bei allen Prozessen die ATPase- Aktivität von Hsp70 essentiell für die Funktion des Proteins. Charakteristisch für das Hsp70-System ist die Kontrolle der Aktivität durch Ko-Chaperone (Hsp40; DnaJ), wobei durch gezielte Modulation der ATPase-Aktivität das Gleichgewicht zwischen Substratbindung und Freisetzung beeinflußt wird (Bukau und Horwich, 1998).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). It has been shown that proteins can only be passed through membranes in the unfolded or partially folded state (Hannavy et al., 1993). During the translocation process in organelles, representatives of the Hsp70 family are primarily involved in the development and stabilization on the cytosolic side, as well as in the refolding on the organelle side (Hauke and Schatz, 1997). The ATPase activity of Hsp70 is essential for the function of the protein in all processes. The control of the activity by co-chaperones (Hsp40; DnaJ) is characteristic of the Hsp70 system, the balance between substrate binding and release being influenced by targeted modulation of the ATPase activity (Bukau and Horwich, 1998).
• Hsp90• Hsp90
Mit etwa 1% des löslichen Proteins im eukaryontischen Cytosol stellt Hsp90 eines der am stärksten exprimierten Proteine dar (Welch und Feramisco, 1982). Die Vertreter dieser Familie agieren vorwiegend in multimeren Komplexen, wobei sie eine Vielzahl von wichtigen Signal- transduktionsproteinen mit nativähnlichen Strukturen erkennen. Durch die Bindung an Hsp90 und seine Partnerproteine werden diese Strukturen stabilisiert und so die Bindung von Liganden an die Signalproteine erleichtert. So können die Substrate ihre aktive Konformation erlangen (Sullivan et al., 1997; Bohen et al., 1995; Buchner, 1999).With about 1% of the soluble protein in the eukaryotic cytosol, 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• HsplOO
In jüngster Zeit zeichneten sich besonders die HsplOO Chaperone durch ihre Fähigkeit in Zusammenarbeit mit den Hsp70 Chaperonen bereits gebildete Aggregate wieder aufzulösen aus (Parsell et al., 1994; Golloubinoff et al., 1999; Mogk et al., 1999). Während ihre Hauptaufgabe die Vermittlung von Thermotoleranz zu sein scheint (Schirmer et al, 1994; Kruger et al., 1994), vermitteln einige Vertreter wie ClpA und ClpB zusammen mit der Protease-Untereinheit ClpP den proteolytischen Abbau von Proteinen (Gottesman et al., 1997).Recently, the 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).
• sHsps• sHsps
Die fünfte Klasse von Chaperonen, die kleinen Hitzeschockproteine (sHsps) stellen eine sehr divergente Familie von Hitzeschockproteinen dar, die in fast allen Organismen gefunden werden. Namensgebend für diese Familie von Chaperonen ist ihr relativ geringes monomeres Molekulargewicht von 15-40 kDa. In der Zelle existieren sHsps aber meist als hocholigomere Komplexe mit bis zu 50 Untereinheiten woraus Molekülmassen von 125 kDa bis zu 2 MDa beobachtet wurden (Spectof et al., 1971; Arrigo et al., 1988; Andreasi-Bassi et al., 1995; Ehrnsperger et al., 1997). In vitro können sHsps wie die anderen Chaperone die Aggregation von Proteinen unterdrücken (Horwitz, 1992; Jakob et al., 1993; Merck et al, 1993; Jakob und Buchner, 1994, Lee et al., 1995; Ehrnsperger et al., 1997b). Dabei binden sHsps bis zu ein Substratmolekül je Untereinheit und zeigen somit eine höhere Effizienz als das Modellchaperon GroEL (Jaenicke und Creighton, 1993; Ganea und Harding, 1995; Lee et al., 1997; Ehrnsperger et al, 1998a). Unter Streßbedingungen verhindert die Bindung von nicht nativem Protein an sHsps die irreversible Aggregation der Proteine. Durch die Bindung an sHsps werden die Proteine in einem löslichem faltungskompetentem Zustand gehalten. Nach der Wiederherstellung von physiologische Bedingungen kann das nicht native Protein von ATP -abhängigen Chaperonen wie Hsp70 aus dem Komplex mit sHsp gelöst und reaktiviert werden.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. In the cell, however, 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). Like other chaperones, 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.
• Isomerasen• isomerases
Als Isomerasen kommen für das erfindungsgemäße Verfahren beispielsweise Faltungskatalysatoren aus der Klasse der Peptidyl-prolyl-cis/trans Isomerasen und Vertreter der Disulfidisomera- sen.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.
Faltungshelferproteine, die in gleicher oder ähnlicher Weise funktionieren wie die oben beschriebenen Faltungshelferproteine, werden ebenfalls von der vorliegenden Erfindung umfaßt.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.
Besonders bevorzugt ist das erfindungsgemäße Verfahren, wenn der Stamm mit verschiedenen Vektoren transformiert wurde, wobei sich die Vektoren mindestens darin unterscheiden, daß die darin enthaltenen Gene für unterschiedliche Helferproteine kodieren. Auf diese Weise können verschiedene Helferproteine, die für die in vitro Synthese des jeweiligen Zielproteins wichtig sind, in einem Lysat hergestellt werden.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. In this way, various helper proteins that are important for the in vitro synthesis of the respective target protein can be produced in one lysate.
Weiterhin ist erfindungsgemäß bevorzugt, daß der Stamm, welcher geeignet ist für die Gewinnung von in vitro Translations-Lysaten, zusätzlich wenigstens eine der folgenden Eigenschaften besitzt: RNAse-arm oder -defizient, Exonuklease-arm oder - defizient, Protease-arm oder defi- zient.It is further preferred according to the invention that 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.
Eine Ausführungsform der Erfindung beinhaltet das erfindungsgemäße Verfahren, wobei das Lysat in der Weise gewonnen wird, daß zusätzlich zu den Helferproteinen alle Komponenten in dem Lysat enthalten sind, welche für eine in vitro Translation oder für eine in vitro Transkription/Translation eines Zielproteins erforderlich sind. Für eine in vitro Translation oder für eine in vitro Transkription/Translation eines Zielproteins sind mindestens die folgenden Komponenten erforderlich: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:
• Ribosomen• ribosomes
• Aminoacyl tRNA-Synthasen• Aminoacyl tRNA synthases
• Initiationsfaktoren• Initiation factors
• Elongationsfaktoren• Elongation factors
• Terminationsfaktoren• Termination factors
• Enzyme, die zu einer Regeneration von ATP, GTP, UTP und CTP ausgehend von einem eingesetzten primären Energiedonor nötig sind. Solche primären Energiedonoren sind beispielsweise Acetylphosphat, Kreatinphosphat, Phosphoenolpyruvat, Pyruvat, Glucose oder weitere dem Fachmann bekannte mögliche Substrate, die direkt oder über mehrere enzymkatalysierte Zwischenschritte umgesetzt werden, so daß Moleküle mit einer energiereichen Phosphatbindung entstehen, welche diese Phosphatgruppe dann wieder auf ein Nukleotidmonophosphat oder ein Nukleotiddiphosphat übertragen können.• Enzymes that are necessary for the regeneration of ATP, GTP, UTP and CTP based on the primary energy donor used. 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.
Gegenstand der vorliegenden Erfindung ist somit auch ein Lysat enthaltend Helferproteine, wobei dieses Lysat erhältlich ist durch das erfindungsgemäße Verfahren. Prinzipiell sind auch andere Verfahren denkbar, mit dem das erfindungsgemäße Lysat erhalten werden kann, z. B. Verfahren, bei denen die Promotoren der in den Stämmen natürlich vorkommenden Helferproteingene verändert werden, so daß eine größere Menge an dem Helferprotein gebildet wird. Eine weitere Methode ist die Transformation von einem Stamm mit einem DNS Stück, welches das co- dierte Helferprotein enthält und welches in das Genom des Stammes ein oder mehrmals integriert wird, um dann von diesem Stamm bei der Zellteilung mit amplifiziert zu werden. Jedes Lysat, daß die gleichen Eigenschaften aufweist, wie das Lysat, welches erhältlich ist durch das erfindungsgemäße Verfahren, ist von der vorliegenden Erfindung umfaßt.The present invention thus also relates to a lysate containing helper proteins, this lysate being obtainable by the process according to the invention. In principle, other methods are also conceivable with which 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.
Erfindungsgemäß bevorzugt ist das. oben beschriebene Lysat, wobei mindestens zwei verschiedene Helferproteine enthalten sind,According to the invention, the lysate described above is preferred, at least two different helper proteins being contained,
Von der Erfindung umfaßt sind auch Lysate enthaltend im wesentlichen ein Helferprotein.The invention also includes lysates essentially containing a helper protein.
Besonders bevorzugt ist das erfindungsgemäße Lysat wobei die Helferproteine ausgewählt werden aus der folgenden Gruppe:The lysate according to the invention is particularly preferred, the helper proteins being selected from the following group:
- Helferproteine des Dnak Systems (DnaK, DnaJ und/oder GrpE)- Helper proteins of the Dnak system (DnaK, DnaJ and / or GrpE)
- Helferproteine des GroE-Systems (GroEL, GroES) Chaperonine- Helper proteins of the GroE system (GroEL, GroES) chaperonins
- Proteindisulfidisomerase,Protein disulfide isomerase,
- Trigger-Faktor und- trigger factor and
- Prolyl-cis-trans Isomerase.- Prolyl-cis-trans isomerase.
Als ganz besonders vorteilhaft können sich Verschnitte erweisen aus verschiedenen erfindungsgemäßen Lysaten. Auf diese Weise könnte die Anzahl der Helferproteine und deren Konzentration für die jeweilige in vitro Translation bzw. in vitro Transkription und Translation des Zielproteins optimiert werden.Blends from various lysates according to the invention can prove to be very particularly advantageous. In this way, 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.
Eine bevorzugte Ausfuhrungsform sind Verschnitt aus einem oder mehreren erfindungsgemäßen Lysaten mit einem Lysat enthaltend alle Komponenten, welche für eine in vitro Translation oder für eine in vitro Transkription/Translation erforderlich sind.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.
Des weiteren ist Gegenstand der Erfindung ein Stamm, welcher geeignet ist für die Gewinnung von in vitro Translations-Lysaten, der mit einem Vektor enthaltend ein oder mehrere für ein oder mehrere Helferproteine kodierende Gene transformiert wurde.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.
Gegenstand der vorliegenden Erfindung ist auch die Verwendung eines erfindungsgemäßen Lysates oder einem erfindungsgemäßen Verschnitt zur in vitro Translation bzw. zur in vitro Transkription/Translation. Des weiteren umfaßt die Erfindung die Verwendung eines erfindungsgemäßen Lysates oder einem erfindungsgemäßen Verschnitt in einem CECF oder CFCF Reaktor. Eine derartige Versuchsanordnung ist in den Prinzipien der "continuous exchange cell- free" (CECF), bzw. der "continuous flow cell-free" (CFCF) Proteinsynthese realisiert (US 5,478,730; EPA 0 593 757; EPA 0 312 612; Baranov & Spirin (1993) Meth. Enzym.217, 123-142). CECF-Reaktoren bestehen aus mindestens zwei diskreten Kammern, welche durch eine poröse Membranen gegeneinander abgetrennt sind. Durch diese poröse Grenzfläche werden die hochmolekularen Komponenten in der Reaktionskammer zurückgehalten, während niedermolekulare Komponenten zwischen Reaktionskammer und Versorgungskammer ausgetauscht werden . Bei CFCF- Verfahren wird eine Versorgungslösung direkt in die Reaktionskammer gepumpt und die Reaktionsendprodukte werden durch eine oder mehrere Ultrafiltrationsmembranen aus dem Reaktionskompartiment gepresst. Entsprechende Reaktorenprinzipien wurden bei der "continuous exchange cell-free" (CECF), bzw. der "continuous flow cell-free" (CFCF) Proteinsynthese realisiert (US 5,478,730; EPA 0 593 757; EPA 0 312 612; Baranov & Spirin (1993) Meth. Enzym. 217, 123-142).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. In 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).
Überraschenderweise konnte durch die Zugabe der Helferproteine auch eine deutliche Steigerung der Expressionsrate der Zielproteine erzielt werden.Surprisingly, the expression of the target proteins could also be increased significantly by adding the helper proteins.
Erfindungsgemäß können die Zielproteine alle Arten von pro- und eukaryontischen Proteinen und auch archaeale Proteine sein. Problematisch in in vitro Transkriptions/Translationssystemen war bisher insbesondere die Expression von sekretorischen Proteinen und Membranproteinen, besonders dann wenn keine Faltungshelferproteine in ausreichender Menge vorhanden sind. Die erfolgreiche Expression von Lipoproteinen und membranständigen Proteinen wurde im Stand der Technik zwar beschrieben, war aber deutlichen Limitationen unterworfen (Hupa und Ploegh, 1997; Falk et al., 1997). Das erfindungsgemäße Verfahren kann sich insbesondere für die Expression von Lipoproteinen und membranständigen Proteinen bzw. sekretorischen Proteinen als Zielprotein eignen, da Faltungshelferproteine durch die Koexpression in ausreichender Menge zur Verfügung gestellt werden.According to the invention, 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.
Des weiteren erwies sich als überraschender Vorteil, daß durch Zugabe der erfindungsgemäßen Lysate zu einem zellfreien in vivo Translationssystem aktive Telomerase hergestellt werden konnte. Bisher konnte weder in prokaryontischen Zellen, noch in zellfreien prokaryontischen Lysaten aktive Telomerase exprimiert werden. Somit ist auch Gegenstand der vorliegenden Erfindung die Verwendung eines erfindungsgemäßen Lysates oder eines erfindungsgemäßen Verschnittes zur in vitro Translation bzw. zur in vitro Transkription/Translation von Telomerase. Die zellfreie in vitro Expression von Telomerase mit prokaryontischen Lysaten konnte erfindungsgemäß durch die Zugabe eines erfindungsgemäßen Lysates enthaltend die Helferproteine DnaK und DnaJ zu einem herkömmlich hergestellten E. coli Extrakt erreicht werden. Durch die Zugabe dieses erfindungsgemäßen Lysates wurde die Aggregation der ungefalteten katalytischen Untereinheit der Telomerase verhindert und die Rekonstitution mit der RNA Komponente hTR zur aktiven Telomerase ermöglicht. Damit besteht aufgrund der vorliegenden Erfindung erstmals die Möglichkeit, aktive und reine Telomerase kostengünstig herzustellen.Furthermore, it turned out to be a surprising advantage that active telomerase 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. coli extract. By adding this lysate according to the invention, the aggregation of the unfolded catalytic subunit of the telomerase was prevented and the reconstitution with the RNA component hTR to the active telomerase was made possible. This is the first time that the present invention makes it possible to produce active and pure telomerase inexpensively.
Da die Telomerase in allen Eukaryonten von der Hefe bis zum Menschen exprimiert wird, ist die Analyse der „reinen" Telomerase schwierig, da prinzipiell immer Kofaktoren aus den Expressionssystemen zusätzlich vorhanden sind.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.
Da in E. coli die meisten posttranslationalen Modifikationen eukaryontischer Zellen nicht vorhanden sind, besteht nun die Möglichkeit, in vitro synthetisierte Telomerase beispielsweise gezielt mit Kinasen zu modifizieren und dadurch die Wirkungsweise und die Einflüsse dieser Modifikationen zu untersuchen.Since most post-translational modifications of eukaryotic cells are not present in E. coli, it is now possible to specifically modify in vitro synthesized telomerase with kinases, for example, and thereby to investigate the mode of action and the effects of these modifications.
Bisher waren auch die Einführung von unnatürlichen Aminosäuren zur Struktur und Funktionsanalyse, oder gezielten post-translationalen Modifikationen Liu J.-P. (1999) Faseb J. 13, 2091, (z. B. Phosphorylierungen) sind in zellulären Expressionssystemen nur eingeschränkt möglich. Durch die zellfreie Synthese der Telomerase beispielsweise stehen nun alle Möglichkeiten zum Einbau unnatürlicher Aminosäuren offen, wie beispielsweise der Einbau von 15N oder 13C markierten Aminosäuren für NMR Untersuchungen, Seleno-markierten Aminosäuren zur röntgen- kristallographischen Analytik, Fluoreszenz- oder Spin-markierten Aminosäuren (Hohsaka et al. (1999) J. Am. Chem. Soc. 121, 12194) zur Untersuchung von Bindungsmechanismen.So far, the introduction of unnatural amino acids for structure and functional analysis, or targeted post-translational modifications Liu J.-P. (1999) Faseb J. 13, 2091, (e.g. phosphorylations) are only possible to a limited extent in cellular expression systems. The cell-free synthesis of telomerase, for example, now opens up all possibilities for incorporating unnatural amino acids, such as the incorporation of 15 N or 13 C labeled amino acids for NMR investigations, seleno-labeled amino acids for X-ray crystallographic analysis, fluorescence or spin-labeled amino acids (Hohsaka et al. (1999) J. Am. Chem. Soc. 121, 12194) for the investigation of binding mechanisms.
Figurenbeschreibungfigure description
Figur 1:Figure 1:
Telomeraseanteil im Pellet beziehungsweise im Überstand des Zentrifugates der Reaktionsprodukte erhalten mit und ohne Zugabe von Helferproteinen.Telomerase content in the pellet or in the supernatant of the centrifugate of the reaction products obtained with and without the addition of helper proteins.
Figur 2:Figure 2:
Anteil eines gelösten Fusionsproteins in Abhängigkeit von der Menge zugegebener Helferproteine. Figur 3:Proportion of a dissolved fusion protein depending on the amount of helper proteins added. Figure 3:
Löslicher Telomeraseanteü in E. coli Lysaten aus zwei verschiedenen Herstellungen in flüssigem oder lyophilisertem Zustand mit und ohne Zugabe von Helferproteinen.Soluble telomerase in E. coli lysates from two different preparations in liquid or lyophilized state with and without the addition of helper proteins.
Figur 4:Figure 4:
Einfluß der Zugabe einzelner Helferproteine zum Lysat auf den löslichen Telomeraseanteü.Influence of the addition of individual helper proteins to the lysate on the soluble telomerase content.
Figur 5:Figure 5:
Einfluß der Zugabe von DnaK und DnaJ mit und ohne GrpE auf den löslichen Telomeraseanteü.Influence of the addition of DnaK and DnaJ with and without GrpE on the soluble telomerase content.
Figur 6:Figure 6:
Einfluß von Helferproteinen des DnaK Systems auf die Aktivität des grün fluoreszierenden ProteinsInfluence of helper proteins of the DnaK system on the activity of the green fluorescent protein
(GFP)(GFP)
Figur 7:Figure 7:
Erhöhung der Synthesemenge von Telomerase in Abhängigkeit von der Zugabe der Helferproteine.Increasing the amount of synthesis of telomerase depending on the addition of the helper proteins.
Figur 8:Figure 8:
Löslicher Telomeraseanteü in Abhängigkeit vom Einsatz von Lysaten aus den ZeUen transformiert mit den DnaK/DnaJ/GrpE SystemSoluble Telomeraseeanteü depending on the use of lysates from the ZeUen transformed with the DnaK / DnaJ / GrpE system
Figur 9:Figure 9:
Löslicher Telomeraseanteü in Lysaten aus dem nicht-transfor ierten A19 Stamm, welche mit 25% bzw. 50% Lysat aus dem mit einem Plasmid codierend für die Proteine aus den DnaK System transformierten A19 Stamm verschnitten wurden.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.
Figur 10:Figure 10:
Coomassie gefärbtes SDS-Gel einer zeü-freien Expression von Rhodanese (35 kDa); Spur 1 und SpurCoomassie stained SDS gel of a zeolite-free expression of Rhodanese (35 kDa); Track 1 and track
2: Ohne Zugabe von RTS GroEL/ES Lysat, Spur 3 und Spur 4: Mit Zugabe von RTS GroEL/ES Lysat.2: Without adding RTS GroEL / ES lysate, lane 3 and lane 4: With adding RTS GroEL / ES lysate.
In Spur 1 und 3 sind jeweüs die Überstandfraktionen, in Spur 2 und 4 die Niederschlagsfraktionen aufgetragen. Figur 11:Lane 1 and 3 each show the supernatant fractions, lane 2 and 4 the precipitation fractions. Figure 11:
Gesamtaktivität der zell-frei exprimierten Rhodanese in Abhängigkeit von der Zugabe des Helferprotein enthaltenden Lysates; Säule 1: Expression ohne Zugabe von GroEL/ES-Lysat; Säule 2: Expression mit Zugabe von GroEL/ES-Lysat.Total activity of the cell-free expressed Rhodanese as a function of the addition of the lysate containing helper protein; Column 1: Expression without addition of GroEL / ES lysate; Column 2: Expression with addition of GroEL / ES lysate.
Die folgenden Beispiele erläutern die Erfindung weiter.The following examples further illustrate the invention.
A. Verwendete ReaktionskomponentenA. Reaction components used
1. Plasmide:1. Plasmids:
pIVEX2.3-GFP: Das Gen für das Green Fluorescent Protein aus Aequoria victoria (Prasher et al. (1992) Gene 111, 229) wurde über die Ncol SchnittsteUe in den ρIVEX2.3 Vektor (Röche Diagnostics GmbH Mannheim, Germany) Moniert.pIVEX2.3-GFP: The gene for the green fluorescent protein from Aequoria victoria (Prasher et al. (1992) Gene 111, 229) was cloned into the ρIVEX2.3 vector (Röche Diagnostics GmbH Mannheim, Germany) via the Ncol interface.
pIVEX2.4b-Mal-Epo: Aus pMAL-p2 (New England Biolabs, Beverley, MA, USA) wurde das Gen für das Maltose bindende Protein isoliert und in den Vektor pIVEX2.4b Woniert. Hinter dieses Gen wurde das Gen für das humane Erythropoietin (Jacobs et al. (1985) Nature 313, 806) ohne die Signalsequenz Woniert, wobei pIVEX2.4b-Mal-Epo entstand.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.
pIVEX2 ,4bNde-hTERT: Das Gen für die katalytische Untereinheit der humanen Telomerase (Autexier C. et al. (1996) EMBO Journal. 15, 5928) wurde in den ρIVEX2.4bNde Vektor über die Nde 1 Schnittstelle einkloniert, wobei ρIVEX2.4bNde-hTERT entstand.pIVEX2, 4bNde-hTERT: The gene for the catalytic subunit of human telomerase (Autexier C. et al. (1996) EMBO Journal. 15, 5928) was cloned into the ρIVEX2.4bNde vector via the Nde 1 interface, where ρIVEX2.4bNde -hTERT was created.
p]NEX2.4-Rhodanese: Das bovine mitochondriale Rhodanese Gen (Miller D.M et al. (1991), J. Biol Chem. 266, 4686) wurde über die Nco I Schnittstelle in den pIVEX2.4- Vektor Moniert, wobei pIVEX2.4-Rhodanese entstand.p] 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.
2. Helferprotein-Plasmide2. Helper protein plasmids
DRDKIG welches für die Proteine Dna-K, Dna-J und GrpE codiert (Dale GE et al. (1 94) Protein Eng. 7, 925), sowie gereinigtes Dna-K, Dna-J und GrpE Protein wurde von Dr. H. Schönfeld Hoffmann- La Röche Ltd., Basel Schweiz erhalten. pREP4-groESL welches für die Proteine Gro-EL und Gro-ES codiert wurde von P. Caspers erhalten (Caspers et al. ( 1994) Cell Mol. Biol.40, 635-44). Gereinigtes GroEL und GroES Protein wurde von Dr. H. Schönfeld Hoffmann- La Röche Ltd., Basel Schweiz erhalten.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.
3. E. coli S30 Lysat3. E. coli S30 lysate
Das Lysat wurde mit einem E. coli A19-Stamm nach dem Verfahren nach Zubay (Annu. Rev. Genet. (1973) 7, 267) präpariert.The lysate was prepared with an E. coli A19 strain using the Zubay method (Annu. Rev. Genet. (1973) 7, 267).
B. Beispiele Beispiel 1B. Examples Example 1
Beispiel la: Einfluss von Helferproteinen auf die Löslichkeit der TelomeraseExample la: Influence of helper proteins on the solubility of telomerase
Das prVEX2.4bNde-hTERT Plasmid wurde im bakterieUen in vitro Expressionssystem mit und ohne Zugabe von je 1 μM der Helferproteine DnaK, DnaJ und GrpE eingesetzt. Für die Expression wurde der Rapid Translation System RTS 500 E. coli circular template Kit (Röche Diagnostics GmbH) verwendet. Die Helferproteine wurden in gereinigter Form zugesetzt. Die Reaktionsprodukte wurden anschließend für 2 min bei lOOOOxg zenfrifugiert. Das resultierende Peüet und der Überstand wurden in SDS-Probenpuffer aufgenommen und auf ein SDS-Gel aufgetragen. Das SDS-Gel wurde mittels Westernblot analysiert. Die Mengen an detektierten Protein sind in Figur 1 zu sehen.The prVEX2.4bNde-hTERT plasmid was used in the bacterial in vitro expression system with and without the addition of 1 μM 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.
Ergebnis: Es zeigte sich, dass im Falle der Zugabe von Helferproteinen ein wesentlich höherer Anteü gelöster Telomerase (Überstandsfraktion) vorliegt.Result: It was found that when helper proteins were added, there was a significantly higher proportion of dissolved telomerase (supernatant fraction).
Beispiel 1b: Einfluss von Helferproteinen des DnaK Systems auf die Löslichkeit eines Fusionsproteins bestehend aus Maltose-Bindeprotein und ErythropoietinExample 1b: Influence of helper proteins of the DnaK system on the solubility of a fusion protein consisting of maltose binding protein and erythropoietin
Das Fusionsprotein wurde vom Expressionsvektor pIVEX2.4b-Mal-Epo im bakteriellen in vitro Expressionssystem mit und ohne Zugabe von je lμM der Helferproteine DnaK, GrpE und DnaJ (analog zu Beispiel la) synthetisiert. Die Reaktionsprodukte wurden anschließend für 2 min bei lOOOOxg zenfrifugiert. Das resultierende PeUet und der Überstand wurden in SDS-Probenpuffer aufgenommen und auf ein SDS-Gel aufgetragen. Das SDS-Gel wurde mittels Westernblot analysiert. Ergebnis: Es zeigte sich, dass im Falle der Zugabe steigender Mengen von Helferproteinen ein steigender Anteü gelösten Fusionsproteins (Überstandsfraktion=Supernatant) vorliegt (Figur 2).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 μM 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. The SDS gel was analyzed by Western blot. Result: It was found that when increasing amounts of helper proteins were added, there was an increasing amount of dissolved fusion protein (supernatant fraction = supernatant) (FIG. 2).
Beispiel 2: Einfluss von Helferproteinen bei verschiedenen Lysatpräparationen und Lysatlyo- philisation Wie in Beispiel 1 wurden E. coli Lysate aus 2 verschiedenen Herstellungen in flüssigem oder lyophüi- siertem Zustand mit und ohne Zugabe von je 1 μM der Helferproteine DnaK, DnaJ und GrpE in einer Telomerase Expression eingesetzt.Example 2: Influence of helper proteins in different lysate preparations and lysate lyophilization As in Example 1, E. coli lysates from 2 different preparations were in a liquid or lyophilized state with and without the addition of 1 μM of the helper proteins DnaK, DnaJ and GrpE in one Telomerase expression used.
Ergebnis: Bei beiden Lysat Herstellungen war ein ähnlich positiver Effekt für die Helferprotein Substitution festzustellen. Das lyophilisierte Lysat zeigte einen geringeren löslichen Telomerase Anteü. Auch hier brachte die Helferprotein Zugabe eine gesteigerte Löslichkeit (Figur 3).Result: A similar positive effect for the helper protein substitution was found in both lysate preparations. The lyophilized lysate showed a lower soluble telomerase content. The helper protein addition also brought about increased solubility here (FIG. 3).
Beispiel 3: Zugabe einzelner Helferproteine zum LysatExample 3: Addition of individual helper proteins to the lysate
Es wurde in diesem Beispiel nicht das ganze DnaK System bestehend aus DnaK, DnaJ und GrpE sondern jeweÜs nur die gereinigten Einzelkomponenten in 1 μM Konzentration zugesetzt. Die Analyse war analog zu Beispiel 1.In this example, not the entire DnaK system consisting of DnaK, DnaJ and GrpE was added, but only the cleaned individual components in 1 μM concentration. The analysis was analogous to Example 1.
Ergebnis: DnaJ und GrpE hatten keinen positiven Einfluß auf die Löslichkeit der Telomerase, während DnaK einen geringen, aber reproduzierbar positiven Effekt hatte (Figur 4).Result: DnaJ and GrpE had no positive influence on the solubility of the telomerase, while DnaK had a slight, but reproducibly positive effect (FIG. 4).
Beispiel 4: Ein Gemisch aus DnaK und DnaJ ist ausreichendExample 4: A mixture of DnaK and DnaJ is sufficient
Ein Gemisch aus DnaK und DnaJ mit und ohne Zugabe von GrpE wurde wie in Beispiel 1 getestet.A mixture of DnaK and DnaJ with and without the addition of GrpE was tested as in Example 1.
Ergebnis: Das Gemisch aus DnaK und DnaJ hat den gleichen Effekt wie das Gesamtgemisch aus allen 3 Komponenten (Figur 5).Result: The mixture of DnaK and DnaJ has the same effect as the total mixture of all 3 components (Figure 5).
Beispiel 5: Einfluss von Helferproteinen des DnaK Systems auf die Aktivität des Grün fluoreszierenden Proteins (GFP)Example 5: Influence of helper proteins of the DnaK system on the activity of the green fluorescent protein (GFP)
Wüdtyp GFP wurde ohne den zur Faltung nötigen Sauerstoff analog zu Beispiel 4 exprimiert indem das Reaktionsgefäß aus dem RTS 500 Kit bis oben aufgefüllt wurde. Nach Beenden der Reaktion wurde das Reaktionsprodukt in ein offenes Gefäß pipettiert und in Gegenwart von Luft-Sauerstoff für 24 Stunden im Kühlschrank gelagert. In dieser Zeit kann der korrekt gefaltete Anteü des GFP Proteins oxidieren und so den Fluorophor ausbüden. Die Aktivität des GFP Proteins wurde dann anhand der Fluoreszenz gemessen.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.
Ergebnis: Die Aktivität von GFP steigt durch Zugabe eines Gemisches aus DnaK und DnaJ (Figur 6).Result: The activity of GFP increases by adding a mixture of DnaK and DnaJ (FIG. 6).
Beispiel 6: Erhöhung der Synthesemenge von Telomerase in Abhängigkeit von der Zugabe von HelferproteinenExample 6: Increasing the amount of synthesis of telomerase depending on the addition of helper proteins
Analog zu Beispiel 4 wurden Mengen von je 1 μM, 2 μM und 3 μM der beiden Helferproteine DnaK und DnaJ in der Telomerase Expression eingesetzt. Die Reaktionsprodukte wurden nun jedoch bei lOO.OOOxg für 30 min zentrifugiert und die Fraktionen im Westernblot analysiert.Analogously to Example 4, amounts of 1 μM, 2 μM and 3 μM 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.
Ergebnis: Während bei 1 μM des Gemisches noch 40 % unlöslicher Telomerase vorhanden war, sank der Anteü unlöslicher Telomerase bei 2 μM des Gemisches auf 8 % und bei 3 μM auf < 1 %.Result: While 40% insoluble telomerase was still present at 1 μM of the mixture, the amount of insoluble telomerase dropped to 8% at 2 μM and <1% at 3 μM.
Die Gesamtmenge an synthetisierter Telomerase stieg bei aUen Ansätzen mit Helferprotein deutlich an. Be dem Ansatz mit 3 μM DnaK/DnaJ war der Anstieg sogar mehr als 50 % (Figur 7).The total amount of telomerase synthesized increased significantly in the case of outside preparations with helper protein. In the approach with 3 μM DnaK / DnaJ, the increase was even more than 50% (FIG. 7).
Beispiel 7: Messung der Aktivität von rekonstituierter Telomerase in Abhängigkeit von Helferproteinen.Example 7: Measurement of the activity of reconstituted telomerase as a function of helper proteins.
Telomerase wurde in Gegenwart von je 0 μM, 2 μM und 10 μM an DnaK und DnaJ exprimiert. Anschließend wurden die Ansätze mit der RNA Komponente rekonstituiert und ein Aktivitätstest mit dem Telo TAGGGG Telomerase PCR ELISA (Röche Diagnostics GmbH) angesetzt. Die Rekonstitution der Telomerase erfolgte nach der Prozedur von Weinrich S. L. et al. (1997) Nature Genet. 17, 498.Telomerase was expressed in the presence of 0 μM, 2 μM and 10 μM 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.
Als Negativkontrolle wurden die Helferproteine vor dem Einsatz in der in vitro Proteinsynthese zunächst für 30 min bei 70 °C hitzebehandelt. Tabelle 1:As a negative control, the helper proteins were first heat-treated at 70 ° C. for 30 min before being used in the in vitro protein synthesis. Table 1:
Ergebnis: Mit Helferproteinen konnte die Aktivität im Vergleich zu dem Ansatz ohne Helferproteine um mehr als das zehnfache gesteigert werden. Die hitzebehandelten Helferproteine waren hingegen völlig inaktiv.Result: With helper proteins, the activity could be increased more than tenfold compared to the approach without helper proteins. The heat-treated helper proteins, however, were completely inactive.
Beispiel 8: Herstellung von Helferprotein produzierenden Stämmen.Example 8: Preparation of helper protein producing strains.
Der Stamm A19 und der Stamm Xl-Blue wurden mit Plasmiden (s. unter A.) transformiert, die entweder die Helferproteine aus dem DnaK/DnaJ/GrpE System oder aus dem GroEL/ES System hinter einem IPTG induzierbaren Promotor enthalten. Der Stamm A19 besitzt eine Mutation mit Rnase I Gen, während der Stamm Xl-Blue eine Defizienz in Protease-Genen besitzt.The 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.
Die transformierten ZeUen wurden auf LB Medium angezüchtet und bei einer optischen Dichte von 1,0 gemessen bei 600 nm Wellenlänge für 30 min mit IPTG (Endkonzentration bis zu 1 mM) induziert.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).
Aus diesen Bakterien wurde ein Lysat für die in vitro Translation entsprechend der Prozedur von Zu- bay G ( 1973) Annu Rev. Genet. 7, 267 hergestellt. Nach Auftrennung der Lysate auf SDS-Gelen und Anfärbung mit Coomassie Brüliant Blue konnte gezeigt werden, dass alle transformierten Stämme je- weüs die entsprechenden Proteine aus dem DnaK/DnaJ/GrpE System oder dem GroEL/ES System exprimieren.A lysate for in vitro translation was made from these bacteria in accordance with the procedure of Zubub G (1973) Annu Rev. Genet. 7, 267. After the lysates had been separated on SDS gels and stained with Coomassie Brüliant Blue, it could be shown that all the transformed strains each express the corresponding proteins from the DnaK / DnaJ / GrpE system or the GroEL / ES system.
Beispiel 9a: Einsatz von Lysaten aus den Zellen transformiert mit dem DnaK/DnaJ/GrpE SystemExample 9a: Use of lysates from the cells transformed with the DnaK / DnaJ / GrpE system
Die Lysate aus den Zellen transformiert mit dem DnaK/DnaJ/GrpE System wurden anschließend zur in vitro Translation mit dem Telomerase Gen eingesetzt. Im Vergleich dazu wurden die Lysate aus den untransformierten Stämmen verwendet. Es konnte gezeigt werden, dass mit dem Lysat aus dem IPTG induzierten transformierten Stämmen im Gegensatz zu dem untransformierten Stämmen zu 100% lösliche Telomerase exprimiert werden konnte (Figur 8).The lysates from the cells transformed with the DnaK / DnaJ / GrpE system were then used for in vitro translation with the telomerase gene. In comparison, 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).
Beispiel 9b:Example 9b
Es konnte gezeigt werden, dass mit dem Lysat aus dem IPTG induzierten transformierten Stämmen im Gegensatz zu dem untransformierten Stämmen zu 100% lösliche Telomerase exprimiert werden konnte.It could be shown that, in contrast to the untransformed strains, 100% soluble telomerase could be expressed with the lysate from the transformed strains induced by the IPTG.
Ergebnis: Bereits 25 % des Helferprotein enthaltenden Lysates reicht aus, um die Löslichkeit der Telomerase auf 90 % zu erhöhen. Mit 50 % dieses Lysates entsteht vollkommen lösliche Telomerase (Figur 9).Result: 25% of the lysate containing helper protein is sufficient to increase the solubility of the telomerase to 90%. Fully soluble telomerase is formed with 50% of this lysate (FIG. 9).
Beispiel 9c: Einsatz von Lysaten, präpariert aus den Zellen, welche mit pREP4-groESL transformiert wurdenExample 9c: Use of lysates prepared from the cells which were transformed with pREP4-groESL
In einem bakterieUen in vitro Expressionssystem (Rapid Translation System RTS 500 E. coli HY Kit, Röche Diagnostics GmbH) wurde bovine Rhodanese durch Verwendung des pINEX2.4-Rhodanese- Plasmids exprimiert (24 h, 30°C), wobei die Expression einmal ohne Zugabe von transformiertem Lysat (Bedingungen wie in der Produktbeschreibung des Herstellers angegeben), das andere Mal unter Zugabe von 50% eines Lysates (aus ZeUen, welche mit pREP4-groESL-Plasmid transformiert worden waren und durch Inkubation GroEL und GroES überexprimiert hatten) durchgeführt wurde. Die Reaktionsgemische wurden anschließend für 5 Min. bei 10.000 xg zentrifügiert, das resultierende Pellet und der Überstand in SDS-Probenpuffer aufgenommen, auf einem SDS-Gel aufgetrennt und mit Coomassie-Blue angefärbt (s. Figur 10).In a bacterieUen in vitro expression system (Rapid Translation System RTS 500 E. coli HY Kit, Röche Diagnostics GmbH), 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).
Ergebnis: Bereits 50 % des Helferprotein enthaltenden Lysates reicht aus, um die LösUchkeit der Rhodanese auf 90 % zu erhöhen.Result: 50% of the lysate containing helper protein is sufficient to increase the Rhodanese solubility to 90%.
Anschließend wurde untersucht, ob durch die Verwendung des GroEL/ES enthaltenden Lysates neben der Löslichkeit auch die Aktivität der exprimierten Rhodanese verbessert werden konnte, wobei die Enzym-Aktivität der beiden vorangegangenen Reaktionen nach der Methode von Weber, F. und Hager-Hartl, M. (Methode Mol. Biol. (2000), 140, 117) bestimmt wurde. Ergebnis: Auch die Aktivität der Rhodanese steigt mit der Zugabe des Helferprotein (GroEL/ES) enthaltenden Lysates signifikant an (s. Figur 11.) It was then investigated whether the use of the lysate containing GroEL / ES, in addition to the solubility, could also improve the activity of the expressed Rhodanese, the enzyme activity of the two previous reactions using the method of Weber, F. and Hager-Hartl, M (Method Mol. Biol. (2000), 140, 117). Result: The activity of Rhodanese also increases significantly with the addition of the lysate containing helper protein (GroEL / ES) (see FIG. 11).

Claims

Patentansprüche claims
1. Nerfahren zur HersteUung eines Lysates enthaltend Helferproteine, dadurch gekennzeichnet, daß ein Stamm, welcher geeignet ist für die Gewinnung von in vitro Translations-Lysaten, transformiert wird mit einem Vektor enthaltend ein oder mehrere für ein oder mehrere Helferproteine kodierende Gene, daß die Helferproteine in diesem Stamm exprimiert werden und daß das Lysat enthaltend Helferproteine aus diesen Stämmen gewonnen wird.1. Nerfahren for the production of a lysate containing helper proteins, characterized in that 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 that the helper proteins are expressed in this strain and that the lysate containing helper proteins is obtained from these strains.
2. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß der Stamm mit verschiedenen Vektoren transformiert wurde, wobei sich die Vektoren mindestens darin unterscheiden, daß die darin enthaltenen Gene für unterschiedliche Helferproteine kodieren.2. The method according to claim 1, characterized in that the strain has been transformed with different vectors, the vectors differing at least in that the genes contained therein code for different helper proteins.
3. Verfahren gemäß Anspruch 1 oder 2 wobei der Stamm zusätzlich wenigstens eine der folgenden Eigenschaften besitzt: RΝAse-arm oder -defizient, ExonuWease-arm oder - defizient, Protease-arm oder defizient.3. The method according to claim 1 or 2, wherein the strain additionally has at least one of the following properties: poor or deficient in Rase, exonu poor or deficient, deficient in protease or deficient.
4. Verfahren gemäß einem der Ansprüche 1 bis 3, wobei das Lysat in der Weise gewonnen wird, daß zusätzlich aUe Komponenten in dem Lysat enthalten sind, welche für eine in vitro Translation oder für eine in vitro Transkription/Translation erforderlich sind.4. The method according to any one of claims 1 to 3, wherein the lysate is obtained in such a way that additional components are contained in the lysate, which are required for in vitro translation or for in vitro transcription / translation.
5. Lysat enthaltend Helferproteine erhältlich durch ein Verfahren gemäß einem der Ansprüche 1 bis 4.5. lysate containing helper proteins obtainable by a method according to any one of claims 1 to 4.
6. Lysat gemäß Anspruch 5 wobei mindestens zwei verschiedene Helferproteine enthalten sind,6. lysate according to claim 5, wherein at least two different helper proteins are contained,
7. Lysat gemäß Anspruch 5 enthaltend im wesentlichen ein Helferprotein.7. Lysate according to claim 5 containing essentially a helper protein.
8. Lysat gemäß einem der Ansprüche 5 bis 7 wobei die Helferproteine ausgewählt werden aus der folgenden Gruppe:8. Lysate according to one of claims 5 to 7, wherein the helper proteins are selected from the following group:
- Helferproteine des Dnak Systems (DnaK, DnaJ und/oder GrpE)- Helper proteins of the Dnak system (DnaK, DnaJ and / or GrpE)
- Helferproteine des GroE-Systems (GroEL, GroES) Chaperonine Proteindisulfidisomerase,- Helper proteins of the GroE system (GroEL, GroES) chaperonine protein disulfide isomerase,
Trigger-Faktor und - Prolyl-cis-trans Isomerase.Trigger factor and - Prolyl-cis-trans isomerase.
9. Verschnitt aus verschiedenen Lysaten gemäß einem der Ansprüche 7 oder 8.9. waste from different lysates according to one of claims 7 or 8.
10. Verschnitt aus einem oder mehreren Lysaten gemäß einem der Ansprüche 5 bis 8 mit einem Lysat enthaltend alle Komponenten, welche für eine in vitro Translation oder für eine in vitro Transkription/Translation erforderlich sind.10. Blend from one or more lysates according to one of claims 5 to 8 with a lysate containing all components which are required for in vitro translation or for in vitro transcription / translation.
11. Stamm, welcher geeignet ist für die Gewinnung von in vitro Translations-Lysaten, der mit einem Vektor enthaltend ein oder mehrere für ein oder mehrere Helferproteine kodierende Gene transformiert wurde.11. Strain which is suitable for obtaining in vitro translation lysates which has been transformed with a vector containing one or more genes coding for one or more helper proteins.
12. Verwendung eines Lysates gemäß einem der Ansprüche 5 bis 8 oder einem Verschnitt gemäß einem der Ansprüche 9 oder 10 bei der in vitro Translation bzw. zur in vitro Transkription/Translation.12. Use of a lysate according to one of claims 5 to 8 or a blend according to one of claims 9 or 10 in in vitro translation or for in vitro transcription / translation.
13. Verwendung eines Lysates gemäß einem der Ansprüche 5 bis 8 oder einem Verschnitt gemäß einem der Ansprüche 9 oder 10 bei der in vitro Translation bzw. in vitro Transkription von Telomerase.13. Use of a lysate according to one of claims 5 to 8 or a blend according to one of claims 9 or 10 in the in vitro translation or in vitro transcription of telomerase.
14. Verwendung eines Lysates gemäß einem der Ansprüche 5 bis 8 oder einem Verschnitt gemäß einem der Ansprüche 9 oder 10 in einem CECF oder CFCF Reaktor. 14. Use of a lysate according to one of claims 5 to 8 or a blend according to one of claims 9 or 10 in a CECF or CFCF reactor.
1/111/11
DnaK/J DnaK/J DnaK/J DnaK/J DnaK / J DnaK / J DnaK / J DnaK / J
T iö.4 T iö.4
2/112/11
T iß-Z T iß-Z
3/113.11
4/114.11
ohne DnaK/J/GrpE DnaK DnaJ GrpE without DnaK / J / GrpE DnaK DnaJ GrpE
5/115/11
ohne DnaK/J/GrpE DnaK/J without DnaK / J / GrpE DnaK / J
6/116/11
ohne DnaK/J mit DnaK/J without DnaK / J with DnaK / J
7/117/11
8/118/11
XL-blue ohne XL-blue A 19 ohne A 19 +DnaK/J +DnaK/JXL-blue without XL-blue A 19 without A 19 + DnaK / J + DnaK / J
9/119.11
A 19 ohne A 19 +25 %DnaK/J A 19 +50 %DnaK/JA 19 without A 19 +25% DnaK / J A 19 +50% DnaK / J
10/1110/11
11/1111/11
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