https://www.abbs.info
E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(11): 759–766
CN 31-1940/Q
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
—————–
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]