Optimization and High-level Expression of a Functional GST-tagged rHLT-B in Escherichia coli and GM1 Binding Ability of Purified rHLT-B

다국어 초록 (Multilingual Abstract)

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 (glutathion 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.

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