Abstract
The utilization efficiency of a secondary energy source in a cell-free protein synthesis system can be improved by use of a metabolic inhibitor. Oxalate, a potent inhibitor of phophoenolpyruvate synthetase, substantially increased the yield of chloramphenicol acetyltransferase synthesis through the enhanced supply of ATP. Oxalate, at 2.7 mM, increased the synthesis yield by 47% when successive amino acids additions prevent amino acid depletion during protein synthesis. These results suggest that cell-free protein synthesis efficiency could also be improved by disrupting the gene encoding phosphoenolpyruvate synthetase.
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References
Davanloo P, Rosenberg AH, Dunn JJ, Studier FW (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81: 2035–2039.
Jermutus L, Ryabova LA, Pluckthun A (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9: 534–548.
Kigawa T, Muto Y, Yokoyama S (1995) Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis. J. Biomol. NMR 6: 129–134.
Kim DM, Swartz JR (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66: 180–188.
Kim DM, Swartz JR (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotechnol. Prog. 16: 385–390.
Narindrasorasak S, Bridger WA (1978) Probes of the structure of phosphoenolpyruvate synthetase: effects of a transition state analogue on enzyme conformation. Can. J. Biochem. 56: 816–819.
Ohuchi S, Nakano H, Yamane T (1998) In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation. Nucl. Acids Res. 26: 4339–4346.
Shaw WV (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Meth. Enzymol. 43: 737–755.
Stiege W, Erdmann VA (1995) The potentials of the in vitro biosynthesis system. J. Biotechnol. 41: 81–90.
Zollner H (1993) Handbook of Enzyme Inhibitors, 2nd edn. Weinheim: VCH Press, p. 887.
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Kim, DM., Swartz, J.R. Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli. Biotechnology Letters 22, 1537–1542 (2000). https://doi.org/10.1023/A:1005624811710
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DOI: https://doi.org/10.1023/A:1005624811710