Abstract: Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins.cDNA encoding the catalytic subunit of Chinese bovine enterokinase(EKL)was amplified by RT- PCR and overlap PCR,then cloned into the yeast secreted expression plasmid p9K.The plasmid p9K/EKLC was transformed GS115 methylotrophic strain of Pichia pastoris. The high expression strain was obtained by screening monoclones.Secreted expression of recombinant EKLC was attained by methanolinduction and its molecular weight was 43 ku. The high-density fermentation supernatant was condensed by ultrafiltration.After only a single-step purification with SP Sepharose F.F column,the purity of fusion protein was above 98% by HPLC and relactive ability was 2.3 U/L.This purified EKLC demonstrates excellent cleavage activitytowards fusion protein containing EK cleavage site.