WO2004072107A1

WO2004072107A1 - Method of preparing protein as a soluble form in a cell-free protein synthesis system

Info

Publication number: WO2004072107A1
Application number: PCT/KR2004/000302
Authority: WO
Inventors: Sang Hyeon Kang; Won Jae Choi; Hyo Jin Kim; Soo Youn Jun; Ki Young Lee
Original assignee: Dreambiogen Co., Ltd.
Priority date: 2003-02-15
Filing date: 2004-02-13
Publication date: 2004-08-26
Also published as: KR100505744B1; KR20040073874A

Abstract

The present invention relates to a method for producing a soluble protein for the treatment, research, and industrial purpose by a cell-free protein production system which is constructed using a cell extract artificially manipulated. More particularly, the present invention relates to a method for producing an soluble target protein by preparing a cell extract containing a large amount of a folding-related protein factor and using the prepared cell extract for the cell-free protein production system.

Description

METHOD OF PREPARING PROTEIN AS A SOLUBLE FORM IN A CELL-FREE PROTEIN SYNTHESIS SYSTEM

FIELD OF THE INVENTION

The present invention relates to a novel cell-free protein synthesis process for enhanced production of soluble protein, and more specifically, a process of preparing cell extracts for a cell-free protein synthesis system wherein the foreign genes for folding-related factors are incorporated into a host cell, the transformant is cultured for over-expression of the folding-related factors, then cell extract is prepared from this cell culture; and a method of preparing a soluble proteins at high levels using the cell-free protein synthesis system comprising the above cell extract.

According to the method of the present invention, proteins produced in biologically inactive and insoluble forms such as aggregates or inclusion bodies when expressed by conventional cell-based expression methods or the conventional cell-free protein synthesis systems can be successfully produced in a soluble form, whereby the soluble proteins can be prepared in a short time at low cost. The method of the present invention is applicable to therapeutic, research and industrial applications to prepare soluble protein samples. Furthermore, structural analysis of proteins for the development of novel therapeutic agents can be carried out by using the method according to the present invention. BACKGROUND OF THE INVENTION

The Human Genome Project has led to a greater understanding of human genes. The complete sequence is not available yet, but efforts to obtain a high quality sequence having error rates of less than 1 base per ten thousand is continuing. Following complete sequencing of the human genome, we will enter a post-genome era in which the function and structure of proteins encoded by the sequenced genes will be easily researched.

In view of keeping up with the requirements in this post-genome era, bioinformatics technologies can partially reveal the structure and function of proteins encoded by the genes using computer programs. Recently, it became possible to integrate genetic information using computers and also systematically analyze it using various analytical programs. While these techniques have enabled rapid predictions, a comprehensive analysis regarding the structure and function of proteins of interest requires more than just computer-generated predictions.

Until now, therefore, a cell-based direct protein expression method for obtaining protein samples (hereinafter, referred to as "direct expression method") has been the most credible method of gaining greater insight into the function and structure of proteins. If protein analyses can be performed using a combination of direct expression and bioinformatics, more relevant results will be obtained. This means, for example, that potentially useful candidate genes will be selected from source data by bioinformatics, then desired ones of the candidate genes can be expressed directly by the direct expression method to verify the existence of the predicted function. However, the cell-based direct protein expression method has some problems. First, the protein to be used in analysis should be produced in a soluble form. The most common technique for cell-based direct protein expression involves using a microorganism such as Escherichia coli (E. coli). The majority of proteins expressed by this in vivo expression method are, however, produced in an insoluble or aggregate form such as inclusion bodies. Although solubilization/refolding procedures are known to transform these proteins into soluble proteins, these are very time-consuming and often inefficient processes, and also they are not generally applicable to all proteins. It is thus most desirable to obtain soluble proteins directly without the requirement for refolding.

The production of inactive, insoluble proteins during heterologous cell-based expression results from the fact that, owing to the difference between the rate of protein synthesis and the rate of protein folding, interactions occur between hydrophobic residues exposed from the folding intermediate of the protein of interest, thereby aggregates are formed.

In order to obtain soluble proteins in cell-based expression systems, many methods are utilized, for example, protein engineering approaches such as substitution of original amino acids with a different amino acids to improve solubility of proteins; fermentational approaches such as temperature adjustment, pH adjustment and/or addition of additives; fusing approaches whereby the protein of interest is fused to proteins of high solubility; and co-expression approaches whereby foldases such as DsbA or PPIase are simultaneously expressed with the desired protein. In addition, there is another technique involving co-expression with a chaperone family protein such as GroEL/GroES or DnaK/DnaJ/GrpE for the production of the soluble protein of interest. Molecular chaperones are a set of proteins which participate in protein folding and prevent the aggregation of newly synthesized proteins and lead to the correctly folded protein. The most abundant and physiologically important chaperones include the GroEL/GroES chaperone family and the DnaK/DnaJ/GrpE chaperone family. It is generally recognized that the DnaK/DnaJ/GrpE and GroEL/GroES chaperone families have synergistic and partially compensatory roles in assisting protein folding. There are certain cases to which such methods using chaperones have been proven useful, but they have not been able to avoid the fundamental problems involved with using living cells as protein expression platforms.

Therefore, a method is required to produce soluble proteins directly and rapidly while solving the problems concerned with the direct expression method as described above. For this reason, a cell-free protein synthesis system can be considered as an alternative due to its flexibility in manipulating protein folding.

One of the most important problems facing the biotechnology research industry is time constraint; and thus a cell-free protein synthesis system having the ability to produce proteins from genes within 24 hours would greatly increase the efficiency of R&D efforts. If such a technique could be found, the bottleneck in the development of drugs based on structural genetics, which is the preparation of protein samples, could be overcome. Cell-free protein synthesis systems were originally employed as molecular biological tools or used to produce proteins that are toxic to host cells. Recent advances in optimization of reaction conditions and development of new bioreactors have allowed cell-free protein synthesis systems to be applied for industrial purposes (Kim, D.M. et al, Eur. J. Biochem. 239:881-886 (1996); Kigawa, T. et al, FEBS Lett. 442:15-19 (1999)).

Since the cell-free protein synthesis technique has greater flexibility in promoting protein folding compared to cell culture methods, problems such as production of inactive and insoluble proteins may be reduced if this technique is complemented by addition of exogenous elements. Through cell-free protein synthesis, the soluble protein of interest may be obtained without the need for solubilization and refolding, which is one of the objects of the present invention.

In spite of such an advantage in promoting protein folding, production of proteins using conventional cell-free protein synthesis systems frequently results in rapid aggregation of these proteins, although to a lesser extent than in cell-based in vivo expression systems.

To enhance the production of soluble proteins and facilitate folding to active conformation, in conventional cell-free protein synthesis systems, a variety of techniques have been applied to alter the environmental conditions, such as the redox potential. However, these techniques face the serious limitation that they must have no effect on the protein synthesis itself.

In one of the conventional cell-free protein synthesis systems (Offen, B., et al, (2002) Biochemica, No. 1, 20-21; Mattingly, J.R., etal, (2000) Arch. Biochem. Biophys.382: 113-122; Kudlicki, W. et al, (1994) J. Biol. Chem. 269: 16549-16553), purified protein- folding-related factors were added to the reaction mixture in order to enhance the production of soluble proteins. Also, there are methods of adding the purified protein- folding-related factors to refolding buffer after the separation/purification of the protein of interest to enhance the specific activity of the protein. The latter method can be employed for both cell-free protein synthesis systems and cell culture methods. However, both methods require an additional process of purifying the folding-related factors before they are added into the reaction mixture or refolding buffer, and therefore they are time-consuming and costly (Tieman, B., et al, (2001) J. Biol. Chem. 276: 44541- 44550; Wang, J., et al, (1998) Proc. Natl. Acad. Sci. USA 95: 12163-12168; Martin, J., et al, (1991) Nature 352: 36-42; Ewalt, K, etal, (1997) Cell 90: 491-500; Szabo, A, etal, (1994) Proc. Natl. Acad. Sci. USA 91 : 10345-10349).

An effort has been made to improve the cell-free protein synthesis systems by heat stimulation of the cell culture during cell extract preparation in order to induce increased expression of folding-related factors. An increased amount of folding-related factors present in the cell-free protein synthesis system leads to an enhanced production of soluble proteins when compared to the normal cell-free protein synthesis system (Japanese Patent Laid Open No. Hei 7-194374). Even though this process is cost effective, the heat stimulation for over-expression of folding-related factors may have a negative effect on cellular components involved in protein synthesis, and this approach can be used for only to induce production of the folding-related factors inherently present in the cell. Thus, this method has many limitations. Moreover, the expression level of folding-related factors by heat stimulation may be low because it utilizes an inherently inefficient expression system. Many powerful expression systems cannot be subjected to this method. As a result, this method has a demerit that the amount of protein-folding-related factors affected is very small. Furthermore, it is difficult to apply quick stimulation and maintain constant heat stimulation even at the experimental level, thus this method has also a serious limitation of being difficult to apply practically.

SUMMARY OF THE INVENTION

An object of the present invention is to overcome the problems associated with conventional cell-free protein synthesis systems. More specifically, the object of the present invention is to provide a process of preparing cell extracts for a cell-free protein synthesis system wherein the foreign genes for folding-related factors are incorporated into a host cell (hereinafter, sometimes referred to as "transformed cell" or "transformant"), the transformant is cultured for over-expression of the folding-related factors, then cell extract is prepared from this cell culture; and a process of preparing a soluble protein at a high level using the cell-free protein synthesis system comprising the above cell extract.

The inventors of the present invention have found that the use of genetic manipulation to incorporate folding-related factors into a host strain used to prepare a cell extract containing the endogenously over-expressed folding-related factors ensures production of high levels of soluble protein, when compared to conventional cell-free protein synthesis methods wherein the purified folding-related factors prepared by a separate purification step are added exogenously to a cell-free protein synthesis reaction mixture. The present invention was accomplished based upon such finding. Another object of the present invention is to provide a cell-free protein synthesis kit having the cell extract containing the over-expressed protein-folding-related factors as a kit component.

Accordingly, the present invention provides a novel process for production of soluble protein, or an improved cell-free protein synthesis process for production of soluble protein, comprising the steps of:

(A) preparation of cells transformed by genes encoding protein-folding-related factors, so that the transformed cells can express enhanced levels of the protein-folding- related factors;

(B) over-expression of the protein-folding-related factors and preparation of a cell extract for a cell-free protein synthesis system from the transformed cells; and

(C) production of a high level of soluble protein in the cell-free protein synthesis system containing the cell extract containing the over-expressed folding- related factors.

Therefore, the main feature of the present invention for producing soluble proteins or obtaining the enhanced level of soluble protein is that the cell-free protein synthesis is carried out using the cell extract containing the over-expressed protein- folding-related factors. This process, i.e., using genetic manipulation to incorporate the foreign folding-related factors into the host strain to construct a transformant and producing a cell extract containing the over-expressed folding-related factors from the transformant, is a new technique that would be valuable for producing high levels of soluble proteins.

DETAILED DESCRIPTION OF THE INVENTION

The cell-free protein synthesis utilized in the present invention is based on the cell-free protein synthesis system as reported previously (Kim, D.M. et al, Eur. J.

Biochem. 239:881-886 (1996)) which is incorporated herein as a reference; however, some modifications have been made to embody the cell-free protein synthesis system of the present invention. An embodiment of the cell-free protein synthesis system of the present invention is illustrated in EXAMPLE 1. In general, a cell-free protein synthesis mixture includes the DNA (in which case, RNA polymerase also needs to be added) or

RNA of the protein of interest, a cell extract containing the components necessary for protein synthesis, an energy source required for protein synthesis, an ionic buffer, and other additives required for protein synthesis. After mixing the components, the reaction mixture is maintained at an appropriate temperature for protein synthesis (in the range of 25 to 37°C depending on the type of cell used to prepare the cell extract). The incubation is carried out for approximately one hour.

The cell extract required for cell-free protein synthesis includes, as components thereof, ribosomes, enzymes required for protein synthesis, and factors such as initiation factors, elongation factors and termination factors required for transcription and translation, which are known as protein synthesis factors. In general, these components remain in the supernatant when the cell is lysed and centrifuged. Various types of cells may be used to prepare a cell extract, including E. coli, yeast, red blood cells, wheat germ, frog eggs, human cells and insect cells. These cells are cultured, ruptured and centrifuged to obtain the desired components to be added to a cell-free protein synthesis system. This process has already been reported, thus no further explanation of the method is needed for the understanding of persons skilled in the art to which the present invention pertains. An exemplary process is described in EXAMPLE 1.

For the folding-related factors, many types are known, including enzymes such as peptidyl-prolyl cis-trans isomerase and disulfide isomerase, heat shock proteins such as hsplO, hsp60, and hsp70 class proteins, E. co/t-derived molecular chaperones such as GroEL, GroES, DnaK, DnaJ, and GrpE, and polypeptide chain binding proteins such as trigger factors. The most representative protein-folding-related factors are the molecular chaperones. The molecular chaperones include, for example, GroES, GroEL, GrpE, DnaK, DnaJ, heat shock proteins, and so on. The GroES/GroEL chaperone family and DnaK/DnaJ/GrpE chaperone family have been proven as to be effective in promoting protein folding, thus they are preferably applied as folding-related factors in the present invention due to the availability of genetic information thereon. However, it should be understood that the folding-related factors of the present invention are not limited to molecular chaperones. Amino acid sequences of GroES, GroEL, DnaK, DnaJ and GrpE are given in FIGS. 5, 6, 1, 8 and 9, respectively.

Genes encoding protein-folding-related factors can be incorporated into a host cell by standard molecular biology methods, t.e., cloning and transformation. For example, restriction enzymes and ligase can be used to clone the appropriate genes into an expression vector and the host cell can be transformed by this constructed plasmid. The maps of the expression vectors used in the present invention are given in FIGS. 10, 11, 12 and 13, respectively.

A skilled person in the art to which the present invention pertains can easily perform the transformation, over-expression of a target protein and preparation of the cell extract as described above, thus the detailed description thereof is omitted herein.

The content of protein-folding-related factors in the cell extract is in the range of from 1 to 10% (w/w); however, it may vary depending upon various factors such as specific physico-chemical properties of the protein of interest. When preparing the cell extract, the content of protein-folding-related factors can be controlled as follows. For example, in case an increased content of protein-folding-related factors is required, cells are cultured for an extended period of time after induction, and in case a decreased content is required, a normal cell extract deficient in the over-expressed folding-related factors can be mixed with a cell extract including the over-expressed protein-folding- related factors.

Proteins which can be produced by the method of the present invention include, for example, but are not limited to α-, β-, γ-interferon, lipase, erythropoietin (EPO), cytokines, interleukins, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), transforming growth factors (TGFs), thrombopoietin (TPO), and tissue plasminogen activator (TPA). The present invention is particularly suitable for proteins that are not produced in a soluble form or that are produced in a poorly soluble form, when expressed by conventional cell-free protein synthesis systems. For reference, the present invention has an additional advantage: the plasmid encoding protein-folding-related factors is removed during preparation of the cell extract, whereby in vitro expression of the protein-folding-related factors from the corresponding plasmid does not occur. Therefore, the energy source and amino acids added to cell-free protein synthesis mixture are used only for the production of the protein of interest and not the protein-folding-related factors. This serves to enhance the productivity of the protein synthesis system of the present invention.

An example of the preparation of a cell extract containing folding-related factors and the production of soluble protein using the cell extract is illustrated in detail in EXAMPLE 1, and an example of the preparation of a cell extract from a mixture of cell cultures including two strains harboring different folding-related factors, respectively, and the production of soluble protein using the cell extract is illustrated in EXAMPLE 2. Herein, the term "mixture of cell cultures" refers to two or more cell types grown at once, i.e., co-cultivation in a single fermentor. An example of the preparation of a mixture of cell extracts each prepared individually and the production of soluble protein using the mixture is illustrated in EXAMPLE 3. In Examples 1, 2, and 3, EPO was employed as a model protein. EPO is a polypeptide containing many hydrophobic amino acids, and which thus forms aggregates easily. As such, no soluble forms or only very low levels of soluble forms of EPO can be obtained from a cell culture or conventional cell-free protein synthesis system.

The present invention is applicable to therapeutic, industrial and research purposes concerned with obtaining soluble forms of protein. Additionally, the present invention can be used as a tool for producing soluble forms of protein for the purpose of assaying functions of newly discovered genes. The present invention provides a kit for expressing and producing soluble proteins. The kit comprises a reaction mixture, containing all the necessary components required for cell-free protein synthesis as well as a cell extract containing the over-expressed folding-related factors, in an appropriate container. Thus, a researcher performs only the addition of the gene of interest and incubation of the resulting kit at an appropriate temperature to obtain the soluble form of the protein of interest. A person skilled in the art to which the present invention pertains can easily understand and reproduce such a kit, thus the detailed description thereof is omitted herein. The cell-free protein synthesis kit according to the present invention can be used to produce soluble proteins at low cost, with reduced time and labor. A number of genes can be expressed in a short period of time and therefore the method and kit of the present invention are appropriate for performing functional analyses of a large number of genes.

A method for co-expression of a gene of interest together with genes for folding-related factors in cell-free protein synthesis reaction could be considered as an alternative to the method of the present invention, but this co-expression in a single incubation may lead to decreased production of the protein of interest due to co- expression of folding-related factors with the protein of interest, and this would be inefficient compared to the method of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and IB show electrophoresis gels, using fluorescent scanning to visualize EPO proteins produced by 3 -hour incubations using a cell extract containing protein folding-related factor according to an embodiment of the present invention, and using a conventional cell-free protein synthesis system, respectively;

FIGS. 2 A and 2B show electrophoresis gels, using fluorescent scanning to visualize EPO proteins produced by 24-hour incubations using a cell extract containing protein folding-related factor according to an embodiment of the present invention, and using a conventional cell-free protein synthesis system, respectively;

FIGS. 3 A and 3B show electrophoresis gels, using fluorescent scanning to visualize EPO proteins produced by 3- and 24-hour incubations using a cell extract containing protein folding-related factor which was prepared from a mixed cell culture of two different types of transformed cells, according to another embodiment of the present invention, and by a conventional cell-free protein synthesis system, respectively;

FIGS. 4 A and 4B show electrophoresis gels, using fluorescent scanning to visualize EPO proteins produced by 3- and 24-hour incubations using a cell extract containing protein folding-related factor which was prepared by mixing individual cell extracts according to still another embodiment of the present invention, and by a conventional cell-free protein synthesis system, respectively;

Figure 5 is the amino acid sequence of protein-folding-related factor, GroEL;

Figure 6 is the amino acid sequence of protein-folding-related factor, GroES;

Figure 7 is the amino acid sequence of protein-folding-related factor, DnaK; Figure 8 is the amino acid sequence of protein-folding-related factor, DnaJ;

Figure 9 is the amino acid sequence of protein-folding-related factor, GrpE;

Figure 10 is the map of expression vector T7-SL3 encoding protein-folding- related factors, GroEL/GroES;

Figure 11 is the map of expression vector T7-KJE3 encoding protein-folding- related factors, DnaK/DnaJ/GrpE;

Figure 12 is the map of expression vector bad-SLl encoding protein-folding- related factors, GroEL/GroES;

Figure 13 is the map of expression vector bad-KJEl encoding protein-folding- related factors, DnaK/DnaJ/GrpE.

EXAMPLES

The following examples have used EPO as a model protein to show that the present invention can effectively produce the soluble form of this protein, and it should be recognized that EPO is only one of many possible proteins that the present invention can be used to produce. Also, the GroES/GroEL chaperone family or DnaK/DnaJ/GrpE chaperone family is used in the following examples but other folding-related factors are included in the scope of the present invention as disclosed in this specification. The present invention will now be illustrated in more detail by the following examples. However, it will be understood that the present invention is not limited to these specific examples, but can be variously modified as will be obvious to one skilled in the art to which the present invention pertains.

EXAMPLE 1: Preparation of cell extracts containing the over-expressed folding- related factors and production of soluble proteins using the same

PREPARATION OF TRANSFORMANTS

Transformed cells harboring genes of folding-related factors were prepared by standard transformation methods. More specifically, BL21(DE3) as an E. coli strain was used as a host cell and plasmids encoding folding-related factors were obtained from Olivier Fayet (Analytical Biochemistry 254:150-152 (1997)). Commercially available vectors (e.g., Takara product code number 3340) can also be used instead as a vector for expression of chaperone. GroES/GroEL chaperone family or DnaK/DnaJ/GrpE chaperone family was used as the folding-related factors and their amino acid sequences are disclosed in FIGS. 5 ~ 9.

The plasmid encoding GroES/GroEL chaperone family (T7-SL3 plasmid: refer to Analytical Biochemistry 254:150-152 (1997)) is represented in FIG. 10. The cell extract prepared from the transformant containing GroES/GroEL chaperone family derived from T7-SL3 plasmid was named as "S30GroEl".

In FIG. 11, represented is the plasmid encoding DnaK/DnaJ/GrpE chaperone family (T7-KJE3 plasmid: refer to Analytical Biochemistry 254:150-152 (1997)). The cell extract prepared from the transformant containing DnaK/DnaJ/GrpE chaperone family derived from T7-KJE3 plasmid was named as "S30Dnal". In FIG. 12, represented is the plasmid encoding GroES/GroEL chaperone family (bad-SLl plasmid: refer to Analytical Biochemistry 254:150-152 (1997)). The cell extract prepared from the transformant containing GroES/GroEL chaperone family derived from bad-SLl was named as "S30GroE2".

In FIG. 13, represented is the plasmid encoding DnaK/DnaJ/GrpE chaperone family (bad-KJEl plasmid: refer to Analytical Biochemistry 254:150-152 (1997)). The cell extract prepared from the transformant containing DnaK/DnaJ/GrpE chaperone family derived from bad-KJEl was named as "S30Dna2".

These transformants can be readily reproduced by one skilled in the field to which the present invention pertains; and as such they were not deposited for application of the present invention.

Preparation of cell extracts

The cell extract containing the over-expressed folding-related factors and protein-synthesis-related components was prepared according to the following procedure. The preparation procedure of normal cell extract deficient for the over- expressed folding-related factors is different from that of the present invention in that antibiotics are not required in the culture medium of the normal cells, because no plasmids are present in the normal cells and no induction step for expression of folding- related factors is required.

The transformants harboring genes of folding-related factors were cultured in

LB media containing the appropriate antibiotics (spectinomycin for T7-SL3 and T7- KJE3, kanamycin for bad-SLl and bad-KJEl) respectively at 37°C for 7-8 hours, then reinoculated into 100 ml of LB medium containing the above antibiotics and cultured overnight. 100 ml of that cell culture was reinoculated into 2 L of 2 x TB media (16 g/L bacto-tryptone; 10 g/L yeast extract; 5 g/L NaCl) and cultured at 37°C in a shaking incubator at 850 rpm.

The expression of T7 RNA polymerase and folding-related factors was induced when the cell density (OD₆oo) reached 0.6. For the transformants containing plasmids with a T7 promoter (T7-SL3 and T7-KJE3), Isopropyl-β-D-galactoside (IPTG) was added to a final concentration of 1 mM to induce expression of folding-related factors, and T7 RNA polymerase was induced simultaneously. For the transformants containing plasmids with a bad promoter (bad-SLl and bad-KJEl), IPTG was added to a final concentration of 1 mM along with arabinose at a final concentration of 0.2%(w/v), to induce expression of T7 RNA polymerase and folding-related factors. In this case, the IPTG induces expression of T7 RNA polymerase and the arabinose induces expression of folding-related factors in the bad-promoter system.

After induction, cells were further cultured until OD₆oo reached 4-5, centrifuged at 6,000 rpm at 4°C for 12 min, and washed three times with a washing buffer (10 mM Tris-acetate, 14 mM magnesium acetate, 60 mM calcium acetate, and 10 mM 2- mercaptoethanol, pH 8.2). After the final centrifugation, the cell pellet was resuspended in 30 ml of suspension buffer (10 mM Tris-acetate, 14 mM magnesium acetate, 60 mM potassium acetate, and 6 mM 2-mercaptoethanol, pH 8.2). The cell resuspension was centrifuged at 16,000 g at 4°C for 30 min to collect the cells and 1.27 ml of S30 buffer (10 mM Tris-acetate, 14 mM Magnesium acetate, 60 mM Potassium acetate, and 10 mM 2-mercaptoethanol, pH 8.2) per 1 g of wet cell weight was used to resuspend the cells.

French press was used at 770 psi to disrupt the cells. The resultant cell extract was centrifuged at 30,000 g for 30 min at 4°C. The supernatant was collected and recentrifuged under the same conditions to carefully collect the fraction of the supernatant below the lipid layer. 3 ml of preincubation buffer (293 mM Tris-acetate, 2 mM magnesium acetate, 10.5 mM ATP, 84 mM creatine phosphate, 44 mM 2- mercaptoethanol, 0.04 mM each amino acids, and 7 units/ml creatine kinase, pH 8.2) was added to each 10 ml of collected supernatant and preincubated at 37°C for 80 min to destroy endogenous mRNA contained in the cell extract. After the preincubation, the preincubated solution was dialyzed four times with 500 ml of S30 buffer at 4°C for 45 min to remove small molecular weight materials and other extraneous materials such as inorganic phosphate which are inhibitory of protein production. After dialysis, the dialyzed cell extract was centrifuged at 4,000 g for 10 min at 4°C to obtain the supernatant which was then aliquoted and stored in liquid nitrogen. The cell extract thus prepared was used in the cell-free protein synthesis reaction as below.

Cell-free protein synthesis

The cell-free protein synthesis reaction was performed based on the method reported by Kim et al (Eur. J. Biochem. 239:881-886 (1996)), with some modifications made thereto. These modifications include using BL21(DE3) strain or its transformants instead of the A- 19 strain, and expressing T7 RNA polymerase in a cell culture prior to preparing the cell extract, whereby the addition of exogenous T7 RNA polymerase to the cell-free protein synthesis reaction mixture became unnecessary. For reference, A- 19 strain cannot express T7 RNA polymerase. The concentration of ions in the cell-free protein synthesis reaction was optimized for the production of soluble proteins. Also, polyethylene glycol was removed from the list of additives because it was found to be unsuitable for the production of soluble proteins.

The reaction mixture for cell-free protein synthesis is as follows: 57 mM Hepes/KOH (pH 8.2), 1.2 mM ATP, 0.85 mM each of GTP, UTP and CTP, 1.7 mM dithiothreitol, 150 mM potassium glutamate, 80 mM ammonium acetate, 16 mM magnesium acetate, 0.17 mg/mL E. coli total tRNA mixture, 34 mg/mL 1-5-formyl- 5,6,7,8-tetrahydrofolic acid, 0.3 U/ml Creatine kinase, 0.5 mM each of amino acids, 6.7 μg/mL circular plasmid (containing the ΕPO gene and a T7 promoter, but ΕPO mRNA can be used instead of the circular plasmid), 28 mM creatine phosphate, 0.6 mM cAMP, and 33% (v/v) cell extract. The reaction mixture was incubated at 30°C for 3 or 24 hours. After completion of the incubation, 5 μl of the incubation mixture was taken as a sample for analysis of the total translated protein (i.e., insoluble and soluble forms of the protein) by SDS-PAGΕ. The remaining reaction mixture was centrifuged at 11,000 g for 10 min to separate the soluble and insoluble fraction. 5 μl samples were taken from the supernatant for analysis of the soluble form of the protein.

Analysis of translated protein

To label the translated protein, FluoroTect^®GreenL_ys in vitro labeling System obtained from Promega (the principle behind this method is to label the side chain of lysine which is incorporated into the polypeptide of the protein of interest and thus render the protein detectable by fluorescence scanning) was used according to the manufacturer's instructions to detect the translated protein. SDS-PAGE and fluorescence scanning was employed to analyze the soluble model protein (i.e., EPO) and the results are shown in FIGS. 1 and 2.

FIGS. 1(A) and (B) show EPO produced after 3 hours of incubation, separated by SDS-PAGE and then detected by fluorescence scanning.

- (A) Lane 1: Total translated EPO obtained from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (A) Lane 2: Soluble translated EPO obtained by centrifugation and collection of the supernatant in a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (A) Lane 3: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30GroEl" as a cell extract.

- (A) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant in the cell-free protein synthesis system utilizing "S30GroEl" as a cell extract.

- (A) Lane 5: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30GroE2" as a cell extract. - (A) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant in the cell-free protein synthesis system utilizing "S30GroE2" as a cell extract.

- (B) Lane 1: Total translated EPO obtained from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (B) Lane 2: Soluble translated EPO obtained by centrifugation and collection of the supernatant from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (B) Lane 3: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Dnal" as a cell extract.

- (B) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Dnal" as a cell extract.

- (B) Lane 5: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Dna2" as a cell extract.

- (B) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Dna2" as a cell extract. As shown from the results, the ratio of soluble protein to total protein synthesized when folding-related factors were present in the translation environment is significantly higher than in the conventional cell-free protein synthesis system. The majority of proteins were expressed in soluble form in all 3 -hour incubations but the amount of soluble protein was greatest when "S30Dnal" was used as the cell extract.

Figures 2(A) and (B) show EPO produced after 24 hours of incubation, separated by SDS-PAGE and then detected by fluorescence scanning.

- (B) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Dna2" as a cell extract. The soluble protein ratio was greatest when cell extracts containing folding- related factors were used. When compared to the results of FIG. 1 in which the reaction was carried out for 3 hours, the results of FIG. 2 show that the amount of soluble protein generally decreases when the reaction is carried out for 24 hours. Despite this fact, it was shown that, in the case of using "S30Dnal" as a cell extract, there is increased production of soluble protein even over a 24-hour reaction.

EXAMPLE 2: Preparation of cell extracts from a mixture of cell cultures including two strains harboring different folding-related factor groups respectively and production of soluble protein using the same

This experiment was carried out in the same manner as in EXAMPLE 1 except for the type of cell extract used for the cell-free protein synthesis reaction. The cell extract was prepared according to the same procedure as in EXAMPLE 1 with one modification. Two strains harboring different folding-related factor groups, respectively, i.e., GroES/GroEL and DnaK/DnaJ/GrpE, were inoculated at the same initial cell population and cultured together to generate a mixed cell culture. The mixed cell culture was used for preparation of a cell extract.

BL21(DE3) transformant harboring T7-SL3 plasmid encoding GroES/GroEL chaperone family and BL21(DE3) transformant harboring T7-KJE3 plasmid encoding DnaK/DnaJ/GrpE chaperone family were cultured together, and the cell extract thus obtained was named as "S30Hybrid GroEl-Dnal". Similarly, BL21(DE3) transformant harboring bad-SLl plasmid encoding GroES/GroEL chaperone family and BL21(DE3) transformant harboring bad-KJEl plasmid encoding DnaK/DnaJ/GrpE chaperone family were cultured together, and the cell extract thus obtained was named as "S30Hybrid GroE2-Dna2". FIG. 3 shows an SDS-PAGE gel of translated EPO visualized by fluorescence scanning. That is, the result shown in FIG. 3 was obtained by separating the translated EPO, produced after performance of the above incubation procedure for (A) 3 hours and (B) 24 hours, respectively, by SDS-PAGE and then detecting the EPO by the fluorescence scanning.

- (A) Lane 1: Total translated EPO from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (A) Lane 2: Soluble translated EPO obtained by centrifugation and collection of the supernatant from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (A) Lane 3: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Hybrid GroEl-Dnal" as a cell extract.

- (A) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Hybrid GroEl- Dnal" as a cell extract.

- (A) Lane 5: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Hybrid GroE2-Dna2" as a cell extract. - (A) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Hybrid GroE2- Dna2" as a cell extract.

- (B) Lane 3: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Hybrid GroEl-Dnal" as a cell extract.

- (B) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Hybrid GroEl- Dnal" as a cell extract.

- (B) Lane 5: Total translated EPO obtained from cell-free protein synthesis system utilizing "S30Hybrid GroE2-Dna2" as a cell extract.

- (B) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Hybrid GroE2- Dna2" as a cell extract. Similarly to the result in EXAMPLE 1, the ratio of soluble protein to total protein synthesized in the presence of the folding-related factors was significantly higher than in a conventional cell-free protein synthesis system. From the result shown in FIG. 2, it is shown that using "S30Hybrid GroEl-Dnal" as a cell extract, containing DnaK/DnaJ/GrpE chaperone family similar to "S30Dnal", yields the greatest amount of soluble protein, in which "S30Dnal" gave the best result in EXAMPLE 1.

EXAMPLE 3: Preparation of a mixture of individual cell extracts derived from separate cell cultures containing different folding-related factor groups and production of soluble proteins using the same

This experiment was generally carried out in the same manner as in EXAMPLE

1 except for the type of cell extract used for the cell-free protein synthesis reaction. Based upon the procedure as described in EXAMPLE 1, cell extracts prepared separately were mixed at a ratio of 1 :1 (v:v) to give a mixed cell extract which was then used as a cell extract for a cell-free protein synthesis reaction.

In the instant EXAMPLE, "S30GroEl" and "S30Dnal" were mixed 1:1 (v:v) and the resultant mixed cell extract was named as "S30Mixed GroEl-Dnal". Likewise, "S30GroE2" and "S30Dna2" were mixed 1:1 (v:v) and the resultant mixed cell extract was named as "S30Mixed GroE2-Dna2". FIG. 4 shows an SDS-PAGE gel of translated EPO visualized by fluorescence scanning. That is, the result shown in FIG. 4 was obtained by separating the translated EPO, produced after performance of the above incubation procedure for (A) 3 hours and (B) 24 hours, respectively, by SDS-PAGE and then detecting the EPO by the fluorescence scanning. - (A) Lane 1: Total translated EPO obtained from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (A) Lane 3: Total translated EPO obtained from cell-free protein synthesis system utilizing "S30Mixed GroEl-Dnal" as a cell extract.

- (A) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Mixed GroEl- Dnal" as a cell extract.

- (A) Lane 5: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S30Mixed GroE2-Dna2" as a cell extract.

- (A) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Mixed GroE2-

Dna2" as a cell extract.

- (B) Lane 1: Total translated EPO obtained from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors. - (B) Lane 2: Soluble translated EPO obtained by centrifugation and collection of the supernatant from a conventional cell-free protein synthesis system utilizing cell extract deficient for the over-expressed folding-related factors.

- (B) Lane 3: Total translated EPO obtained from the cell-free protein synthesis system utilizing "S3 OMixed GroE 1 -Dnal " as a cell extract.

- (B) Lane 4: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Mixed GroEl- Dnal" as a cell extract.

- (B) Lane 5: Total translated EPO obtained from cell-free protein synthesis system utilizing "S30Mixed GroE2-Dna2" as a cell extract.

- (B) Lane 6: Soluble translated EPO obtained by centrifugation and collection of the supernatant from the cell-free protein synthesis system utilizing "S30Mixed GroE2- Dna2" as a cell extract.

Similarly to the results in previous EXAMPLES, the ratio of soluble protein to total protein synthesized in the presence of the folding-related factors was significantly higher than in a conventional cell-free protein synthesis system. From the result shown in FIG. 3, it is also shown that using "S30Mixed GroEl-Dnal" as a cell extract, containing DnaK/DnaJ/GrpE chaperone family similar to "S30Dnal", yields the greatest amount of soluble protein, in which "S30Dnal" gave the best result in EXAMPLE 1. CONCLUSION

According to the method of the present invention, folding-related factors were incorporated into a conventional cell-free protein synthesis reaction to promote the production of soluble forms of translated proteins. A conventional cell-free protein synthesis system was modified to be more suitable for the production of soluble protein. Cell extract was prepared from genetically engineered cells that had been modified to express enhanced levels of protein-folding-related factors to assist the folding of soluble proteins into their proper conformation. The cell-free protein synthesis system containing the over-expressed protein-folding-related factors yielded a significantly higher ratio of soluble versus insoluble proteins.

Cell-free protein synthesis is useful due to its flexibility in manipulating the protein folding environment. In spite of this advantage, production of proteins frequently results in rapid aggregation of these proteins, although to a somewhat lesser extent when compared to cell-based in vivo expression. This aggregation is still problematic for direct functional analysis using cell-free translation mixture without purification. To obtain a higher ratio of soluble proteins, a conventional cell-free protein synthesis system has employed purified folding-related factors. For use of the purified folding-related factors, they need to be prepared by a purification step prior to use in the cell-free protein synthesis system. This is time-consuming and laborious due to additional processes required for the purification of the folding-related factors, or costly due to the cost involved in purchasing the purified folding-related factors. These methods have led to the enhanced production of soluble proteins but at a high cost in terms of funds or labor and time. Meanwhile, in the present invention, the folding-related factors are contained within the cell extract at a high level and therefore no additional folding-related factors need to be added, which means that soluble proteins can be produced at a lower cost. Therefore, the problem of the formation of insoluble proteins by conventional cell- based expression systems can be overcome by use of the present invention following a simple procedure, and at low cost when compared to conventional cell-free protein synthesis systems. For these reasons, the present invention is simpler and more cost efficient for high level production of soluble proteins compared to conventional cell- free protein synthesis systems as well as cell-based expression systems.

Cell-based expression systems have the frequent problem of producing insoluble proteins that are inactive and thus unsuitable for functional and structural analysis of proteins. Even though conventional cell-free protein synthesis systems offer a solution to this problem, the process is time consuming, laborious and costly. The present invention has made improvements to the conventional cell-free protein synthesis systems to produce a high level of soluble proteins at low cost, and therefore this invention can be utilized for the functional and structural analysis of proteins to assign functions to genes.

Up until now, a cell-free protein synthesis system utilizing cell extract containing over-expressed folding-related factors derived endogenously from cells transformed by plasmids encoding the folding-related factors has not been developed.

Therefore this novel cell-free protein synthesis system is anticipated to be suitable for producing soluble proteins. Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered exemplary only, with the scope of particular embodiments of the invention indicated by the following claims.

Claims

1. A method for production of soluble protein comprising the steps of:

(C) production of a high level of soluble protein in the cell-free protein synthesis system containing the cell extract containing the over-expressed folding-related factors.

2. The process according to claim 1, wherein said folding-related factor is one or more in number.

3. The process according to claim 1, wherein said folding-related factor is a chaperone.

4. The process according to claim 3, wherein said chaperone is GroES/GroEL chaperone family or DnaK/DnaJ/GrpE chaperone family.

5. The process according to claim 1, wherein said cell extract is prepared from one or more transformed cells.

6. The process according to claim 1, wherein said protein is selected from the group consisting of α-, β-, γ-interferon, lipase, erythropoietin, cytokines, interleukins, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, thrombopoietin, and tissue plasminogen activator.

7. A cell-free protein synthesis kit comprising the cell extract containing a substantial amount of folding-related factor of claim 1.