Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis.
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.
Ottone
Sophie
S
BioMérieux, R&D New Markers Discovery, Chemin de l'Orme, Marcy l'Etoile 69280, France.
Nguyen
Xuan
X
Bazin
Julien
J
Bérard
Cédric
C
Jimenez
Sandrine
S
Letourneur
Odile
O
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D019149:Bioreactors; D002478:Cells, Cultured; D005285:Fermentation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006509:Hepatitis B / Q000175:diagnosis; D006514:Hepatitis B Surface Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D006514:Hepatitis B Surface Antigens; D011994:Recombinant Proteins