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KMID : 0545120100200030550
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 3 p.550 ~ p.557
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Comparative study on characterization of recombinant B subunit of E. coli heat-labile enterotoxin (rLTB) prepared from E. coli and P. patoris
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Ma Xing-Yuan
Yao Bi
Zheng Wen-Yun
Li Lin-Feng
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Abstract
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Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvant eliciting strong immunoresponse to co-administered antigens. In the research, high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in 5 L fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, though the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a serial of screenings and identifications. Fusion proteins LTB-6× His could be secreted into supernatant of medium after the recombinant P. pastoris were induced by methanol 0.5% (v/v) at 30℃, while E. coli transformants expressed target protein in conclusion body after induced by 1mM IPTG at 37℃. The expression level increased dramatically to 250-300 mg/L supernatant of fermentation in the former while 80-100 mg/L in the latter. The LTB-6× His were purified to 95% by affinity chromatography and characterized by SDS-PAGE and Western-blot. Adjuvant activity of target protein was analyzed by binding ability with GM1 gangliosides. MW of LTB-6× His expressed in P. pastoris demonstrates greater than that in E. coli which equal to expected 11 kDa, which may be resulted from glycosylation by P. pastoris that wound enhance immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB have significant advantages in higher expression level and in adjuvant activity compared to homologous E. coli system.
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KEYWORD
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rLTB, characterization, expression and purification, P.patoris and E.coli
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