Journal of Biotechnology
Volume 61, Issue 3,
13 May 1998
, Pages 199-208
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https://doi.org/10.1016/S0168-1656(98)00033-9Get rights and content
A cell-free protein synthesis system using wheat-germ extract was improved by a novel approach involving selective removal of endogenous phosphatase, which reduces both the duration and the rate of translation by hydrolyzing ATP and GTP, from the translational reaction. Immunodepletion of the phosphatases by the antibodies raised against the major one of the wheat-germ phosphatase isozymes removed 20–40% of ATP-hydrolysis activity from the wheat-germ extract, and thereby prolonged the reaction period of translation. Moreover, the condensation of the phosphate-immunodepleted extract by polyethylene glycol (PEG) precipitation and the addition of copper ions, which was known to inhibit phosphatase and nuclease activity, increased the protein synthesis more than two-fold compared with the reaction using control IgG-treated condensed extract.
Cell-free protein synthesis has been re-evaluated as an alternative protein expression system comparable to the conventional system using living cells. Protein production using the cell-free system can avoid (so) many problems such as aggregation of synthesized protein and cell-lysis caused by expressing a toxic gene product. The cell-free expression system also provides a rapid method of the retrieval of a gene product from a PCR-amplified DNA fragment. Moreover, it allows us to synthesize unique polypeptides site-specifically including unnatural amino acids such as halogenated (Noren et al., 1989), and isotope-labeled amino acids that provide a good prove for structural analyses of a protein (Ludlam et al., 1995; Yabuki et al., 1998).
However, the cell-free protein synthesis system had a crucial drawback; the amount of synthesized protein was quite small in any cell-free systems because of their short reaction period (<1 h). A solution to the drawback has been demonstrated by cell-free continuous flow (CFCF) system (Spirin et al., 1988). In this system, the substrate solution including ATP, GTP and amino acids was continuously supplied to the membrane-immobilized translation mixture, resulting in continuous production of protein for >20 h (Kigawa and Yokoyama, 1991; Endo et al., 1992; Kudlicki et al., 1992). For such a long reaction period, the maintaining of the levels of ATP and GTP in the system was proved to be essential by us (Kawarasaki et al., 1994, Kawarasaki et al., 1995) and others (Kitaoka et al., 1996; Matveev et al., 1996; Yao et al., 1997). The use of the CFCF system in a laboratory-scale protein production is, however, far from convenient because of the need of the special device and low reproducibility.
The conventional batch reaction system still has some advantages in terms of reaction reproducibility, cost, operational convenience and the final concentration of the product. Therefore, the batch system rather than the CFCF system should be improved to achieve a more efficient and convenient protein synthesis system.
In the batch reaction system using wheat-germ extract, the most critical factor in the halt of translation has been shown to be the ATP-degradation, which is caused by endogenous phosphatase (Kawarasaki et al., 1995). The increased phosphatase activity, which occurred in condensation of the extract by polyethylene glycol (PEG) precipitation, significantly reduced a translation longevity due to the degradation of ATP and GTP and accumulation of the liberated inorganic phosphate, though the translation rate was increased 10-fold (Nakano et al., 1996).
In wheat germ, we have already reported that there are at least nine acid-phosphatase (EC. 184.108.40.206) isozymes that dephosphorylate ATP and GTP as substrates, and we isolated six relatively stable isozymes and purified one of them, termed B-4 isozyme, to homogeneity (Kawarasaki et al., 1996). Since five isozymes were reacted with a mouse antibody raised against the B-4 isozyme, these isozymes were thought to be immunochemically similar (Kawarasaki et al., 1996).
Using immunodepletion by means of their immunochemical similarity, we show the selective removal of the phosphatase isozymes from wheat-germ extract, which are capable of producing a larger amount of protein owing to the decreased phosphatase activity.
Materials and methods
Protein A conjugated resin (protein A-sepharose FF) was purchased from Pharmacia Biotech (Uppsala, Sweden). Protein G PLUS-Agarose was purchased from Oncogene Science (USA). Some other chemical compounds were from Wako Pure Chemicals (Osaka, Japan). Rabbit for immunization of the B-4 isozyme was Japanese White (female, 2 kg). Wheat germ was provided by Nippon Flour Mills CO., Ltd. (Atsugi-city, Kanagawa Prefecture, Japan).
Immunodepletion of phosphatase from wheat-germ extract
In our previous paper (Kawarasaki et al., 1996), we analyzed wheat-germ phosphatase isozymes, which were assigned from B-1 to B-9 according to their mobility in native PAGE-activity staining. Among them, six isozymes, from B-4 to B-9, were partially isolated, and an antiserum was raised by a mouse against the B-4 isozyme that was purified to homogeneity. Then using the mouse antiserum, the isolated isozymes were subjected to SDS-PAGE followed by western blotting (Fig. 1A). The antiserum gave a
In the cell-free system using wheat-germ extract, ATP and GTP degradation and phosphate accumulation mainly catalyzed by phosphatase isozymes was shown the most critical bottleneck to accomplish a highly productive protein synthesis system in our previous papers (Kawarasaki et al., 1995; Nakano et al., 1996). Here we demonstrated that a selective removal of the phosphatases from translation system was an effective approach.
The antisera raised against the B-4 isozyme showed a cross reactivity
This study was supported by a grant from Asahi Glass Foundation. We thank Dr S. Sekiguchi, Nippon Flour Mills Co., Ltd., for providing of wheat germ and cap analog.
- W.N. Burnette
"Western Blotting": Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A
- Y. Endo et al.
Production of an enzymatic active protein using a continuous flow cell-free translation system
- Y. Kawarasaki et al.
A long-lived batch reaction system of cell-free protein synthesis
- Y. Kawarasaki et al.
Purification and some properties of wheat germ acid phosphatases
- Y. Kitaoka et al.
Cooperativity of stabilized mRNA and enhanced translation activity in the cell-free system
- W. Kudlicki et al.
High efficiency cell-free synthesis of proteins: Refinement of the coupled transcription/translation system
- S.V. Matveev et al.
Effect of the ATP level on the overall protein biosynthesis rate in a wheat germ cell-free system
Biochim. Biophys. Acta
- H. Nakano et al.
Highly productive cell-free protein synthesis system using condensed wheat-germ extract
- R. Serrano et al.
Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria
J. Biol. Chem.
- Y. Kawarasaki et al.
Prolonged cell-free protein synthesis in a batch system using wheat germ extract
Biosci. Biotech. Biochem.
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Improvement of translation efficiency in an Escherichia coli cell-free protein system using cysteine
2011, Protein Expression and Purification
Citation Excerpt :
For example, amino acid limitation in cell-free protein synthesis can be acute [6,7], particularly in the case of arginine, cysteine, serine and tryptophan, which can be depleted within 1 h . Two approaches have been used to address limited amino acid supply: inhibit enzymatic activities causing substrate instability [9,10], or remove the genes encoding the enzymes responsible for depletion from the source strain used for cell extract preparation) [11,12]. Using the second approach, an E. coli KC6 cell strain was generated containing deletions for the genes encoding arginine decarboxylase (speA), tryptophanase (tnaA), serine deaminases (sdaA and sdaB) and glutamate-cysteine ligase (gshA) [11,12].
Cell-free protein synthesis systems are powerful tools for protein expression, and allow large amounts of specific proteins to be obtained even if these proteins are detrimental to cell survival. In this report we describe the effect of cysteine on cell-free protein synthesis. The addition of cysteine caused a 2.7-fold increase in the level of synthesized glutathione S-transferase (GST). Moreover, the levels of sulfhydryl group reductants, including reduced glutathione and dithiothreitol (DTT), were increased 1.9- and 1.7-fold, respectively, whereas levels of the disulfide dimers, cystine and oxidized glutathione, were suppressed 87% and 66%, respectively. These trends were also observed for green fluorescent protein (GFP) expression. The addition of cysteine competitively reversed the inhibitory effect of cystine on protein expression. These results suggest that the sulfhydryl group in cysteine plays a crucial role in enhancing protein synthesis, and that the addition of excess cysteine could be a convenient and useful method for improving protein expression.
An improved cell-free system for picornavirus synthesis
2007, Journal of Virological Methods
Cell-free synthesis of an infectious virus is an ideal tool for elucidating the mechanism of viral replication and for screening anti-viral drugs. In the present study, the synthesis of Encephalomyocarditis virus (EMCV) from its RNA in HeLa and 293-F cell extracts was enhanced by employing a dialysis system in combination with a ribozyme technology. Although translation and processing of the EMCV polyprotein were not accelerated greatly by the dialysis system, de novo synthesis of viral RNA was enhanced considerably by dialysis, leading to a greater than eight-fold increased titer of synthesized EMCV compared with a conventional batch system. Furthermore, a synthetic EMCV RNA with a hammerhead ribozyme sequence at its 5′-end served as an efficient template for viral synthesis in the dialysis system. Therefore, this system provides opportunities for mutational analyses of EMCV in vitro.
Preparative scale cell-free expression systems: New tools for the large scale preparation of integral membrane proteins for functional and structural studies
Cell-free expression techniques have emerged as promising tools for the production of membrane proteins for structural and functional analysis. Elimination of toxic effects and a variety of options to stabilize the synthesized proteins enable the synthesis of otherwise difficult to obtain proteins. Modifications in the reaction design result in preparative scale production rates of cell-free reactions and yield in milligram amounts of membrane proteins per one millilitre of reaction volume. A diverse selection of detergents can be supplied into the reaction system without inhibitory effects to the translation machinery. This offers the unique opportunity to produce a membrane protein directly into micelles of a detergent of choice. We present detailed protocols for the cell-free production of membrane proteins in different modes and we summarize the current knowledge of this technique. A special emphasize will be on the production of soluble and functionally folded membrane proteins in presence of suitable detergents. In addition, we will highlight the advantages of cell-free expression for the structural analysis of membrane proteins especially by liquid state nuclear magnetic resonance spectroscopy and we will discuss new strategies for structural approaches.
Total amino acid stabilization during cell-free protein synthesis reactions
2006, Journal of Biotechnology
Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75–250% relative to cell-free expression using the control extract.
Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system
2006, Journal of Microbiological Methods
In an E. coli cell-free protein synthesis system, the addition of an inorganic polyphosphate [poly(P)] with polyphosphate:AMP phosphotransferase (PAP), which regenerates AMP to ADP, increased the amount of protein synthesis. The maximum yield of the translation product (green fluorescent protein) in the E. coli cell-free system provided by Roche Diagnostics (RTS-100) was 1.16 mg/ml under the optimum reaction condition, which corresponded to a 5.7-fold of that obtained under the standard reaction condition described in the manufacturer's protocol. Interestingly, poly(P) alone enhanced protein synthesis to some extent. When we added poly(P) to the reaction mixture, ATP was consumed at a faster rate, leading to a rapid accumulation of AMP. By adding both poly(P) and PAP to the reaction mixture, an efficient ATP regeneration reaction derived from AMP occurred and the ATP level was recovered. Since the protein synthesis enhancement by poly(P) was also observed when mRNA was added as the template in the reaction, poly(P) accelerated the translation reaction by directly affecting the translation machinery. This also occurred when we used the Pure-system Classic Mini kit (Post Genome Institute) that contained the minimum requirements (pure enzymes and chemicals) for translation and transcription. We also observed that poly(P) extended the half-life of the mRNA template.
Stabilization of affinity-tagged recombinant protein during/after its production in a cell-free system using wheat-germ extract
2003, Journal of Bioscience and Bioengineering
We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.
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