On-column refolding and purification of transglutaminase from Streptomyces fradiae expressed as inclusion bodies in Escherichia coli.
Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture.
Liu
Xiao-Qiu
XQ
State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Yang
Xiu-Qing
XQ
Xie
Fu-Hong
FH
Song
Li-Ya
LY
Zhang
Guo-Qing
GQ
Qian
Shi-Jun
SJ
D001120:Arginine / Q000494:pharmacology; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D002479:Inclusion Bodies / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D012710:Serum Albumin, Bovine / Q000378:metabolism; D012995:Solubility; D013302:Streptomyces / Q000201:enzymology*; D011503:Transglutaminases / Q000096:biosynthesis / Q000302:isolation & purification*; D014508:Urea / Q000494:pharmacology
D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D014508:Urea; D001120:Arginine; D011503:Transglutaminases; D005990:Glycerol