[High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system].
In order to make a large-scale preparation of(GLP-1A2G)2-HAS (GGH), the double-plamid pPICZalphaB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR to create the recombinant expression plasmid pPICZalphaB-ggh, which was transformed into P. pastoris GS 115/F2 that was integrated by another recombinant expression plasmid pPIC9K-ggh. The immunology method combined with high concentration antibiotic was used to screen recombinant strain P. pastoris GS115/F3 capable of high-level expression of GGH protein. The GGH fusion protein expressed by GS115/F3 increased 49.7% compared with the GS 115/F2 in the expression conditions of 3% methanol inducing 80 h at 30 degrees C. At the same time, the quantitative PCR results showed that GGH gene dose in GS115/F3 increased 26.7% with respect to that of GS 115/F2. Furthermore, the Western blotting experiment indicated that the recombinant GGH possess the two antigenicities of GLP-1 and HSA.
Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China.
Hui
H
Wang
Wenfang
W
Dou
Xiaomei
X
Zhang
Hongyu
H
Xu
Zhenghong
Z
Xu
D005822:Genetic Vectors / Q000235:genetics; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D052216:Glucagon-Like Peptide 1