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Searching for Growth Conditions for Optimized Expression of Recombinant Proteins in Escherichia coli by Using Two‐Dimensional NMR Spectroscopy
Author(s) -
Chae Young Kee,
Kim Seol Hyun
Publication year - 2015
Publication title -
bulletin of the korean chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.237
H-Index - 59
ISSN - 1229-5949
DOI - 10.1002/bkcs.10016
Subject(s) - escherichia coli , sorbitol , trehalose , metabolite , chemistry , biochemistry , yield (engineering) , recombinant dna , isoelectric point , nuclear magnetic resonance spectroscopy , osmoprotectant , metabolome , metabolomics , chromatography , amino acid , enzyme , stereochemistry , materials science , metallurgy , gene , proline
Escherichia coli has been widely used as a host to produce recombinant proteins. However, because of its prokaryotic nature, it often fails to produce eukaryotic proteins in their native or correctly folded forms. To devise a systematic way to find such a condition that produces a large amount of correctly folded proteins, we chose to perturb the composition of intracellular metabolites by imposing different stresses such as the addition of sorbitol, NaCl , pH , and ethanol, and to monitor which metabolites showed the largest differences in the case of successful protein production. We profiled the metabolites under stresses by using two‐dimensional NMR spectroscopy, and identified 39 metabolites after the protein production phase. We found that mild NaCl stress increased the yield of the target protein. Mildly high pH stress also helped in producing more of the target protein, and there is likely a connection between the isoelectric point of the target protein and the pH of medium for a higher yield. Exogenous sorbitol appeared to have been used to produce an osmoprotectant (trehalose) or direct energy source (glucose); thus it is advised that its use should not include isotopic labeling. Mild ethanol stress did not cause any significant change either in the metabolite profile or in the target protein production. We hope this work might shed light on how to improve production of the target protein by adjusting the external environment.