Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx. Tag and a 6xHis. Tag at its 5' terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25degreesC gave twice the amount of recombinant gloshedobin in cytoplasm than at 37degreesC.