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| Expression of Fusion Protein NtA-PACAP in E. coli and Its Purification and Biological Analysis |
| WANG Jing, WU Lu-sheng, CHEN Xiao-jia, DONG Hao-jun, SHEN Wei, ZHANG Ming-yi, HONG An |
| Biomedicine Institute & Cell Biology Department of Jinan University,National Engineering Research Center of Genetic Medicine, Guangdong Provincial,Key Laboratory of Bioengineering Medicine, Guangzhou 510632, China |
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Abstract Objective:To obtain a fusion protein NtA-PACAP, which PACAP38 links with the N-terminal domain of Agrin (NtA), in a recombinant prokaryotic system and to analysis its bioactivity. Methods: The nucleotide sequence encoding fusion NtA-PACAP protein were designed and synthesized according E.coli codon bias, and His-tag was added to the C-terminal, then the sequence cloned into vector pET-3c. And this recombinant plasmids were transformed to E.coli BL21(DE3) for expression by IPTG induction. The targeted protein was got by two-step purification methods, which Anion Exchange Chromatography and then Ni-NTA affinity chromatography. Finally its bioactivity was evaluated by the cell injury repair experiments in vitro. Results:NtA-PACAP protein is obtained successfully. Bioactivity assay result shows that NtA-PACAP could promote PC12 cells proliferation after starvation injury. Conclusion: The fusion NtA-PACAP38 protein is worth of developing to a candidate drug.
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Received: 08 November 2012
Published: 25 March 2013
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