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ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36(7): 513–517 CN 31-1300/Q

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Expression of Recombinant Chinese Bovine Enterokinase Catalytic Subunit in P. pastoris and Its Purification and Characterization

Lei FANG, Qi-Ming SUN, and Zi-Chun HUA*

The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China

Abstract Enterokinase is a tool protease widely utilized in the cleavage of recombinant fusion proteins. cDNA encoding the catalytic subunit of Chinese bovine enterokinase (EK_L) was amplified by PCR and then fused to the 3' end of prepro secretion signal peptide gene of a-mating factor from Saccharomyces cerevisiae to get the a-MF signal-EK_L-His₆ encoding gene by PCR. Then the whole coding sequence was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter and transformed GS115 methylotrophic strain of Pichia pastoris. Secreted expression of recombinant EK_L-His₆ was attained by methanol induction and its molecular weight is 43 kD. Because of the existence of His₆-tag, EK_L-His₆ was easily purified from P. pastoris fermentation supernatant by using Ni²⁺ affinity chromatography and the yield is 5.4 mg per liter of fermentation culture. This purified EK_L-His₆ demonstrates excellent cleavage activity towards fusion protein containing EK cleavage site.

Key words recombinant enterokinase; secreted expression; Ni²⁺ affinity chromatography; fusion protein cleavage

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Received: April 19, 2004 Accepted: May 29, 2004

Abbreviations: EK, enterokinase; EK_L, catalytic subunit of Chinese bovine enterokinase; GST-VAS, GST-vasostatin

The study was supported by a grant from the Teaching and Research Award Program for the Outstanding Young Teachers in Higher Education Institutions of Ministry of Education of China

*Corresponding author: Tel/Fax, 86-25-83324605; E-mail, [email protected]