Hirudin in Pichia pastoris secretion expression of.docVIP

 PAGE \* MERGEFORMAT 15 Hirudin in Pichia pastoris secretion expression of Author: Long indium, Liu Yun, LIU Li, Zong-ren [Keywords:] Pichia pastoris Secretory expression of hirudins in Pichia pastoris [Abstract] AIM: To construct hirudin expression vectors and obtain its secretory expression in Pichia pastoris (P. pastoris). METHODS: Hirudin HV2 and HV2N47K genes were amplified by PCR and the correct genes were fused to pPIC9 α factor signal and subcloned into pPIC9K secretory expression vector. The constructs were transformed into P. pastoris strain GSM1168, stable multicopy integrants were screened on medium containing increasing concentrations of G418. Then the screenedout clones were induced by methanol to express hirudin proteins. The supernatants of expression products were identified by SDSPAGE and Western blot analysis, and its antithrombotic activity was determined too. RESULTS: The expression vectors were successfully constructed, hirudins were expressed in supernatants of P. pastoris, and the expression products possessed antithrombotic activity. CONCLUSION: Hirudins can be expressed secretorily in P. pastoris, which offers a basis for newdrug researches. [Keywords] hirudin; Pichia; secretory expression [Abstract] Objective: To construct hirudin expression vector of Pichia pastoris to obtain recombinant protein in Pichia pastoris secreted expression. Methods: PCR amplification hirudin HV2, HV2N47K gene, the sequence into the correct sequence of yeast gene expression carrier pPIC9α secretion signal downstream, and then subcloned into the high-copy vector pPIC9K, construct expression plasmid pPIC9KHV2, pPIC9KHV2N47K. recombinant plasmid was transformed into host bacteria GSM1168, G418 resistance screening of the stable expression of high copy strain induced expression by methanol as well as the expression products were identified . Results: The successful construction of the hirudin expression vector of Pichia pastoris to obtain hirudin in