Purification and reactiongenicity of human immunodeficiency virus type 2 total external glycoprotein expressed in Pichia Pastoris.
The purification of human immunodeficiency virus type 2 total external glycoprotein gp105 expressed in Pichia pastoris was investigated. Expression conditions were optimized by an orthogonal test. The results from tests of variance analyses showed that the most important parameter for efficient expression of total gp105 in P. pastoris is adequate aeration during methanol induction. The optimum induction conditions for gp105 expression were: more than 85% aeration, induction for 3 days, the initial pH 6.0-7.0 and a final methanol concentration of 1.0%. Under these conditions, the expressed total gp105 was secreted into fermentation broth and reached a yield of 23%, approximately 141 mg/l. Expressed gp105 was isolated and purified by salting out and Sephadex G-100 chromatography and the yield of gp105 was 40%. gp105 was purified to electrophoretic purity and its isoelectric point (pI) was about 5.2 by SDS-PAGE and isoelectrofocusing. The purified gp105 contained approximately 35% carbohydrate, which proved that the expressed gp105 was a glycoprotein. Its N-terminal amino acid was arginine by Dansyl-Cl and the result indicated that expressed gp105 was secreted and cleaved correctly. The results from gp105 ELISA demonstrated that the purified total gp105 showed good reactiongenicity and antigenic specificity.
Zhang
Ying J
YJ
College of Life Science, Jilin University, Changchun 130023, PR China.
Jin
Ning Y
NY
Jin
Hong T
HT
Shen
Jia C
JC
D003001:Cloning, Molecular / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D015686:Gene Products, env / Q000032:analysis* / Q000235:genetics; D015498:HIV-2 / Q000302:isolation & purification*; D006801:Humans; D010843:Pichia / Q000235:genetics* / Q000254:growth & development; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D054299:env Gene Products, Human Immunodeficiency Virus
D015686:Gene Products, env; D054299:env Gene Products, Human Immunodeficiency Virus; C069790:gp105 envelope protein, Human immunodeficiency virus 2