Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide.
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.
Yu
Rong-Jie
RJ
Bio-engineering Institute of Jinan University, Guangzhou 510632, China.
Hong
An
A
Dai
Yun
Y
Gao
Yuan
Y
D001483:Base Sequence; D001671:Biochemistry / Q000379:methods*; D002686:Chitin / Q000737:chemistry; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D047668:Inteins; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000302:isolation & purification*; D009479:Neuropeptides / Q000737:chemistry* / Q000302:isolation & purification*; D018377:Neurotransmitter Agents / Q000737:chemistry* / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA
C495401:ADCYAP1 protein, human; D009414:Nerve Growth Factors; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D010455:Peptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D002686:Chitin; D004229:Dithiothreitol