Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis

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• 作者：Sophie Ottone ; Xuan Nguyen ; Julien Bazin ; Cé ; dric Bé ; rard ; S ; rine Jimenez ; Odile Letourneur
• 关键词：Hepatitis B virus ; Hepatitis B surface antigen ; Pichia pastoris ; Anti-HBs antibody ; Diagnosis ; Immunoassay
• 刊名：Protein Expression and Purification
• 出版年：2007
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：December 2007
• 卷：56
• 期：2
• 页码：177-188
• 文件大小：1233 K

摘要

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.