Gene Cloning and Expression of PACAP and Study of Its Biological Activity
- VernacularTitle:垂体腺苷酸环化酶激活肽基因的合成表达与活性研究
- Author:
Zheyu CHEN
;
Yanfeng CHAI
;
Cheng HE
;
Changlin LU
;
Xiangfu WU
- Publication Type:Journal Article
- Keywords:
pituitary adenylate cyclase activating polypeptide;
gen e cloning;
gene expression;
PC12 cells;
spinal cord neurons
- From:
Progress in Biochemistry and Biophysics
2001;28(2):192-197
- CountryChina
- Language:Chinese
-
Abstract:
In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypep tide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP w as deduced and six partially complementary oligonucleotide fragments were design ed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3 ). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that t he GST-PACAP fusion protein was highly expressed and accumulated to about 30% o f the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could prom ote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.
