Optimization and high-level expression of a functional GST-tagged rHLT-B in Escherichia coli and GM1 binding ability of purified rHLT-B.
The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathione S-transferase) in E. coli BL21 (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GM1-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92% purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.
Ma
Xingyuan
X
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R.China.
Zheng
Wenyun
W
Wang
Tianwen
T
Wei
Dongzhi
D
Ma
Yushu
Y
D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007785:Lactose / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli; D005982:Glutathione Transferase; D007785:Lactose