Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium.

@article{citeulike:827829, abstract = {Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40\% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed.}, address = {Laboratory of Molecular Biotechnology, Graduate School of Bio- and Agro-Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. yamanetu@agr.nagoya-u.ac.jp}, author = {Yamane, T. and Ikeda, Y. and Nagasaka, T. and Nakano, H. }, citeulike-article-id = {827829}, doi = {http://dx.doi.org/10.1021/bp0400238}, issn = {8756-7938}, journal = {Biotechnol Prog}, keywords = {cell-free, expression, protein, system}, number = {2}, pages = {608--613}, posted-at = {2006-09-04 16:19:32}, priority = {2}, title = {Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium.}, url = {http://dx.doi.org/10.1021/bp0400238}, volume = {21}, year = {2005} }

TY - JOUR ID - citeulike:827829 L3 - citeulike-article-id:827829 TI - Enhanced cell-free protein synthesis using a S30 extract from Escherichia coli grown rapidly at 42 degrees C in an amino acid enriched medium. JF - Biotechnol Prog VL - 21 IS - 2 SP - 608 EP - 613 AD - Laboratory of Molecular Biotechnology, Graduate School of Bio- and Agro-Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. yamanetu@agr.nagoya-u.ac.jp SN - 8756-7938 N2 - Growths of Escherichia coli strain A19 were investigated in a 5-L fermentor at 37 and 42 degrees C either in Pratt's medium (a standard medium for cell-free protein synthesis using its S30 extract) or in a casamino acids supplemented Pratt's medium (aa-enriched medium). Specific growth rates in Pratt's medium at 37 and 42 degrees C were 0.77 and 0.46 h(-1), respectively, whereas those in the aa-enriched medium at 37 and 42 degrees C were 0.87 and 1.49 h(-1), respectively. The extent of cell-free chloramphenicol acetyltransferase (CAT) synthesis was compared at 37 degrees C incubation (from a plasmid pK7-CAT) for S30 extracts prepared from the cells cultured in the aa-enriched medium at 37 or 42 degrees C. A 40% increase in CAT synthesis occurred when the 42 degrees C/S30 extract was used as compared with 37 degrees C/S30 extract. CAT and both the light and heavy chains (Lc and Hc) of the Fab fragment of an antibody 6D9 were synthesized at 37 degrees C in the cell-free synthesis in the presence of [(14)C]Leu. Their reaction mixtures were subjected to SDS-PAGE autoradiographic analysis. It was found that most of the synthesized proteins were in the soluble fraction when 42 degrees C/S30 extract was used, suggesting that the 42 degrees C/S30 extract contained greater amounts of various protein folding factors. A dialysis membrane minibioreactor with a reaction volume ca. 0.5 mL was handmade by the authors. The advantages of the minibioreactor are a simple configuration, a low manufacturing cost, and the capability of the dialysis membrane replacement. Increased CAT synthesis was also observed for continuous exchange cell-free (CECF) protein synthesis at 37 degrees C when the 42 degrees C/S30 extract was used in the minibioreactor. Some plausible reasons to give higher protein synthesis activity of the 42 degrees C/S30 extract are discussed. KW - cell-free KW - expression KW - protein KW - system AU - Yamane, T AU - Ikeda, Y AU - Nagasaka, T AU - Nakano, H PY - 2005///r UR - http://dx.doi.org/10.1021/bp0400238 ER -