Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 56, Issue 2, Pages 177-188Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.07.008
Keywords
hepatitis B virus; hepatitis B surface antigen; Pichia pastoris; Anti-HBs antibody; diagnosis; immunoassay
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This study describes the expression in Pichia pastoris of hepatitis B surface antigens (HBsAg) corresponding to the S region of the four major subtypes: adr, adw2, ayr and ayw3 and to the preS2-S region of the two subtypes adr and adw2. The recombinant yeast strains have been selected amongst methanol utilization positive (Mut(+)) or sensitive strains (Mut(s)) and cultivated to high cell density in bioreactor using a short protocol. Our results prove the efficiency of P. pastoris to produce all the major HBsAg subtypes and confirm the ability of the methanol regulated promoter of alcohol oxidase I gene (AOX) to express heterologous protein through phenotype Mut(+) or Mut(s) strains. All these recombinant HBsAg proteins, including subtype ayr, whose production has never been presented, have been highly purified using a short original sequence of steps which includes high-pressure cell disruption associated with detergent treatment, ultrafiltration and immunopurification chromatography using a mAb anti-HBs. The whole process avoids possible alterations of antigenic properties and allows to obtain with high yield, high quality reagents for in vitro diagnosis. (C) 2007 Elsevier Inc. All rights reserved.
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