Abstract: Objective: To establish a Pichia yeast expression system expressing EGF-like domains of thrombomo-dulin domain 2(TM-D2) to obtain recombinant TM-D2 protein. Methods: The nucleotide sequence encoding TM-D2 was amplified by PCR before being ligated into pPICZaB vector. Then TM-D2-pPICZaB vector was constructed and confirmed by PCR and sequencing. After linearization, this recombinant vector was transformed into yeast GS115 by electric shock. Methanol was used to induce the expression of TM-D2, and the TM-D2 was identified by SDS-polyacrylamide gel electrophoresis and Western blotting. Results: Sequencing and PCR revealed that recombinant fragment of TM-D2 with his-tag was cloned into pPICZaB vector, and SDS-PAGE and Western blotting confirmed that TM-D2 protein was present in the supernatant. Conclusions: The recombinant vector TM-D2-pPICZaB is successfully constructed, and TM-D2 is well expressed in Pichia pastoris.

Key words: Thrombomodulin, Recombinant vector, Pichia pastoris