Cloning of PTD-NPY fusion gene and its secretory expression in Pichia pastoris.
- Author:
Yucheng SUN
1
;
Fengqiu ZHOU
;
Jiayu WAN
;
Hongwei GAO
Author Information
1. Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun 130062, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cloning, Molecular;
Gene Fusion;
Genetic Vectors;
genetics;
Neuropeptide Y;
genetics;
Pichia;
genetics;
metabolism;
Polymerase Chain Reaction;
methods;
Rats;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Transduction, Genetic;
tat Gene Products, Human Immunodeficiency Virus;
genetics
- From:
Chinese Journal of Biotechnology
2008;24(9):1620-1624
- CountryChina
- Language:Chinese
-
Abstract:
The PTD-NPY fusion gene derived from HIV-1 TAT protein transduction domain and rat neuropeptide Y was amplified by overlap extension PCR, digested and subcloned into yeast expression vector pPICZ alpha A to construct recombinant expression plasmid pPICZ alpha-PTD-NPY. The cloned PTD-NPY fusion gene was identified by PCR and restriction enzyme digestion and sequenced. The exact recombinant plasmid was linearized by Sac I and integrated by electrotransformation into the genome of Pichia pastoris GS115 cells. Then, these positive recombinant yeast cells were induced by 10 mL/L methanol to express soluble PTD-NPY fusion protein. After 120 h of methanol induction, the SDS-PAGE electrophoresis result indicated PTD-NPY fusion protein was efficiently secreted into the medium. Western blotting analysis proved that the expressed fusion protein had specific NPY binding activity. The successful expression of PTD-NPY fusion protein in Pichia pastoris provided basis for its further application study.
