Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli.
Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.
Kim
Sung-Gun
SG
Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea.
Kweon
Dae-Hyuk
DH
Lee
Dae-Hee
DH
Park
Yong-Cheol
YC
Seo
Jin-Ho
JH
D001407:Bacillus / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005964:Glucosyltransferases / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics; D013997:Time Factors
D018832:Molecular Chaperones; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase