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Cloning and expression of the external‐glycoprotein gene mutant from HIV‐2 in the methylotrophic yeast Pichia pastoris and identification of the glycoprotein
Author(s) -
Zhang Ying J.,
Jin Ning Y.,
Jiang Wen Z.,
Zhu Xue J.,
Shen Jia C.
Publication year - 2001
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20010014
Subject(s) - pichia pastoris , recombinant dna , biology , microbiology and biotechnology , mutant , gene , glycoprotein , polyclonal antibodies , cloning (programming) , pichia , heterologous expression , codon usage bias , expression vector , heterologous , genetics , genome , antibody , computer science , programming language
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.