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ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36(7):
513–517 CN 31-1300/Q
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 (EKL) 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-EKL-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 EKL-His6 was
attained by methanol induction and its molecular weight is 43 kD. Because of
the existence of His6-tag, EKL-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 EKL-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; EKL,
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]