Overproduction of DnaJ in Escherichia coli improves in vivo solubility of the recombinant fish-derived transglutaminase.
The overexpression of red sea bream (Pagrus major) transglutaminase (TGase, E.C. 2.3.2.13) in Escherichia coli mostly leads to the accumulation of biologically inactive enzyme. Although the solubility of the gene products could be improved by cultivation at a lower temperature (26-28 degrees C), most of the synthesized TGase was still in the form of insoluble aggregates. The effects of overproduction of molecular chaperones on the intracellular solubility of newly produced recombinant TGase were examined. The overexpression of dnaK or groES/EL did not improve solubility. However, DnaJ greatly increased the solubility of the recombinant TGase, resulting in active enzyme in the presence of calcium ions. Co-expression of dnaK along with dnaJ further increased the content of soluble TGase. Under our experimental conditions, supplementation with both DnaJ and DnaK elevated the TGase activity in the producer cells by roughly 4-fold, compared with the control strain cultured at 30 degrees C. Thus, we found that DnaJ is important in controlling the solubility of protein overproduced in E. coli.
Food Research & Development Laboratories, Ajinomoto Co., Inc Kanagawa, Japan. flr_yokoyama@tel1.ajinomoto.co.jp
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Yokoyama
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Kikuchi
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Yasueda
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D018832:Molecular Chaperones / Q000096:biosynthesis; D010473:Perciformes / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D013696:Temperature; D011503:Transglutaminases / Q000378:metabolism*
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D011503:Transglutaminases