Cloning and expression of the external-glycoprotein gene mutant from HIV-2 in the methylotrophic yeast Pichia pastoris and identification of the glycoprotein.
To achieve high-level expression of HIV-2(ROD) external glycoprotein gp105 in Pichia pastoris, the gp105 gene mutant tP1, with the 5' non-functional region of the gp105 gene removed, was obtained by PCR amplification and was cloned into secreted expression vector pHILS1. The His(+)Mut(s) recombinant P. pastoris strain was screened by PCR and induced by methanol. SDS/PAGE and Western-blot analyses showed that mutation of the low-usage codon AGG into synonymous codon CGA and the introduction of the optimal codon TTC made P. pastoris overexpress tP1, an 85 kDa heterologous glycoprotein that was secreted into the medium and recognized specifically by HIV-2 polyclonal antibody. The recombinant strain GS115/tP1 had excellent genetic stability in terms of the properties of growth and expression of gp105, and seven out of 58 recombinant stains with a yield of 29% were selected to be used for further purification of gp105.
Zhang
Y J
YJ
College of Life Science, Jilin University, Changchun, 130023, People's Republic of China. yingjiu_cn@sina.com
Jin
N Y
NY
Jiang
W Z
WZ
Zhu
X J
XJ
Shen
J C
JC
D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015498:HIV-2 / Q000235:genetics*; D006639:Histidine / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus
D003062:Codon; D015686:Gene Products, env; D006023:Glycoproteins; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C069790:gp105 envelope protein, Human immunodeficiency virus 2; D006639:Histidine