Expression and purification of soluble HIV-1 envelope glycoprotein gp160 mutant from Saccharomyces cerevisiae.
Here we report the expression of HIV-1 gp160 and its mutated proteins in Saccharomyces cerevisiae. Two strong hydrophobic regions, aa 511-537 and aa 679-703, were predicted by GCG Wisconsin Package software and removed to investigate the solubility of the mutated gp160 (gp160Delta12). The results showed that gp160Delta12 assumes high solubility as to be present in supernatant of cell lysate exclusively. The mutant exists as trimeric form in solutions via some inter-molecule disulfide bonds, which can be associated to monomer with the reduced reaction of DTT. The fermentation procedure was optimized to get high cell density yield and expression level as approximately 10 mg/L. After purification with electro elution, gp160Delta12 was checked as glycosylation form by Endo-H deglycosylating catalysis. The ELISA performed with a panel of human sera suggests that the purified gp160Delta12 shares some determinants with gp120 and gp41, but exposes some distinct epitopes that react with early HIV-infected antibody. Thus, we may provide a novel antigen for immunodetection assay, vaccine candidate, and other relative research purposes.
Yang
Kunyu
K
National Institute of Diagnostics and Vaccine Development in Infectious Disease, School of Life Sciences, Xiamen University, Xiamen, China 361005.
Li
Shaowei
S
He
Fangping
F
Zhang
Jiannan
J
Wei
Minxi
M
Pan
Huirong
H
Yang
Chunyan
C
Xiong
Junhui
J
Zhang
Jun
J
Xia
Ningshao
N
D001483:Base Sequence; D055162:Biocatalysis; D015153:Blotting, Western; D003470:Culture Media; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005285:Fermentation; D019302:HIV Envelope Protein gp160 / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012995:Solubility
D003470:Culture Media; D017931:DNA Primers; D019302:HIV Envelope Protein gp160