[Cloning of PTD-NPY fusion gene and its secretory expression in Pichia pastoris].
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.
Sun
Yucheng
Y
Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun 130062, China.
Zhou
Fengqiu
F
Wan
Jiayu
J
Gao
Hongwei
H
D000818:Animals; D003001:Cloning, Molecular; D050939:Gene Fusion; D005822:Genetic Vectors / Q000235:genetics; D009478:Neuropeptide Y / Q000235:genetics*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D009478:Neuropeptide Y; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus