Secretory expression of hirudins in Pichia pastoris

AIM: To construct hirudin expression vectors and obtain its secretory expression in Pichia pastoris(P. pastoris). METHODS: Hirudin HV2 and HV2-N47K 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 screened-out clones were induced by methanol to express hirudin proteins. The supernatants of expression products were identified by SDS-PAGE 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 new-drug researches.