High-level expression of fusion protein GGH in Pichia pastoris GS115 by constructing a double plasmid co-expression system.
- Author:
Hui WANG
1
;
Wenfang DOU
;
Xiaomei ZHANG
;
Hongyu XU
;
Zhenghong XU
Author Information
1. Laboratory of Pharmaceutical Engineering, School of Medicine and Pharmaceutics, Jiangnan University, Wuxi 214122, China.
- Publication Type:Journal Article
- MeSH:
Genetic Vectors;
genetics;
Glucagon-Like Peptide 1;
biosynthesis;
genetics;
Humans;
Pichia;
genetics;
metabolism;
Plasmids;
genetics;
Polymerase Chain Reaction;
methods;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Serum Albumin;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2011;27(7):983-989
- CountryChina
- Language:Chinese
-
Abstract:
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.
