title: Comparative study on characterization of recombinant B subunit of E. coli heat-labile enterotoxin (rLTB) prepared from E. coli and P. patoris.
abstract: Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-l fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6x His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at 30 degrees C, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at 37 degrees C. The expression level increased dramatically to 250- 300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6x His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GM1 gangliosides. The MW of LTB-6x His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Ma
authors.forename: Bi
authors.initials: B
authors.lastname: Yao
authors.forename: Wenyun
authors.initials: W
authors.lastname: Zheng
authors.forename: Linfeng
authors.initials: L
authors.lastname: Li
mesh_terms: D000276:Adjuvants, Immunologic / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D051147:Ganglioside Galactosyltransferase / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli; D051147:Ganglioside Galactosyltransferase