High-level Expression Of Fusion Protein GGH In Pichia Pastoris GS115 By Constructing A Double Plasmid Co-expression System

Glucagons glucagon-like peptide 1 (GLP -1) has lots of physiological functions, such as provoking insulin biological synthesis and secretion, promoting the proliferation ofβcell in pancreas while inhibiting its apoptosis, suppressing glucagon secretion and so on, which is expected to become the newest treatment for type 2 diabetes drug. GLP-1 could be easily degradated by DPPⅣin vitro, which limited its clinical application. In order to overcome the shortcoming, overlap PCR technology was employed to amplify the fusion gene((GLP-1A2G)2-HSA(GGH)), and the construction of recombinant expression plasmid pPIC9K/GGH was electrotransformated to Pichia pastoris expression system and got a high yield strains Pichia pastoris GS115/F2. The fusion protein GGH not only possessed hpyerglycemic activity and repair of islet function, but also had biological activity after 72h in vivo.In this study, a high throughput screening model was established, which is in situ two-film method. It is a rapid method based on immunology to screen Pichia pastoris transformants expressing high-level protein, in which yeast colonies are lifted from solid culture onto a cellulose acetate film. Then a nitrocellulose film is used to capture the secreted proteins that pass through the cellulose acetate film. The proteins binding on the nitrocellulose film are stained by immunology methods. Finally picked different color colonies for flask culture, it showed that the staining intensity was correlated to the expression level in submerged culture.In order to make a large-scale preparation of GGH, in this paper the double-plamid pPICZαB and pPIC9K co-expression system was introduced into Pichia pastoris GS115. Firstly, the GGH fusion gene was amplified by PCR, and created the recombinant expression plasmid pPICZαB-GGH, which was transformed into P. pastoris GS115/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 GS115/F2 in the expression conditions of 3% methanol inducing 80 h at 30°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 GS115/F2. Furthermore, the western blot result indicated that the recombinant GGH possessed the two antigenicities of GLP-1 and HSA. In 5 L fermentor, when 1% methanol pulsed inducing 80 h, the cell density by OD600 reached 250 and the yield of GGH was about 800 mg/L.