Expression, purification and bioactivity analysis of granulysin
- Author:
Cheng QIAN
1
Author Information
1. Department of Laboratory Diagnosis
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2006;27(8):829-833
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express the granulysin protein in E. coli and to detect its bioactivity after purification. Methods: Using cultured human peripheral blood mononuclear cells as the template, we selectively amplified the fragment coding granulysin by RT-PCR. The fragment was then inserted into the prokaryotic expression vector pET28a (+) for transforming E. coli BL21(DE3) plysS. Fusion protein expression was induced by isopropyl-Beta-D-thiogalactopyranoside (IPTG). After renaturation, the protein was purified by affinity chromatography and its bioactivity was examined by colony-forming unit. Results: We successfully inserted granulysin cDNA fragment into vector pET28a (+) and constructed the expression plasmid pET28a (+)-GNLY. The fusion protein, with a molecular weight of 9000, was obtained through IPTG induction. After renaturation and purification, the recombinant protein was proven to be bioactive by CFU and its cytotoxicity was in a dose-dependent manner. Conclusion: The fusion protein obtained in the present study is bioactive and can be used for further study on its function, mechanism of action and clinical application.
