Induced Secretion Expression of Salivary Apyrase from Culex quinquefasciatus in Pichia pastoris

Salivary Apyrase(ATP diphosphohydrolase) from Culex quinquefasciatus was expressed in Pichia pastoris, which can lay foundation for further studying biological functions of Culex quinquefasciatus Apyrase in saliva.Biological information and RT-PCR technology were adopted for cloning and encoding mature peptide genome sequence of Culex quinquefasciatus Apyrase, it was cloned to downstream alpha-factor signal peptide sequence of Pichia pastoris constitutive secretion expression vector pGAPZ alpha-A. pGAPZa alpha-A-Apyrase recombinant secretion expression vector was constructed. Pichia pastoris GS115 competent cells were electroporated after Bln I linearization treatment on expression vector. Transformants underwent Zeocin resistance screening, colony PCR and SDS-PAGE analysis. pGAPZ alpha-A-Apyrase/ G5115 engineering bacteria secreted and expressed Apyrase recombinant protein at expected size of 60 kD. It was fermented in shake flask for 72 h. The amount of protein expression achieved the highest level.The study proves that Culex quinquefasciatus Apyrase can achieve secretion expression in Pichia pastoris.