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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(11): 759-766 CN 31-1940/Q

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Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

Rong-jie YU, An HONG*, Yun DAI, and Yuan GAO

Bio-engineering Institute of Jinan University, Guangzhou 510632, China

Abstract In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAP-intein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

Key words intein; recombinant human pituitary adenylate cyclase activating peptide (rhPACAP); purification

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Received: July 23, 2004 Accepted: September 17, 2004

This work was supported by the grants from the Guangdong Pro-vincial Science Key Foundation of China (No. 021202) and Guang-dong Provincial Scientific Strategic Special Project of China (No. 2004A10902002)

*Corresponding author: Tel, 86-20-8522-0220; Fax, 86-20-8522-6616; E-mail, [email protected]