Expression, purification of annexin A4-EGFP fusion protein and preliminary study on its apoptosis detection ability
10.16438/j.0513-4870.2017-0340
- VernacularTitle:Annexin A4-EGFP融合蛋白的表达纯化及其凋亡检测能力初探
- Author:
Jing LIU
1
;
Jie WANG
1
;
Zi-chun HUA
1
Author Information
1. School of Life Sciences, Nanjing University, Nanjing 210023, China
- Publication Type:ORIGINAL ARTICLES
- Keywords:
annexin A4;
prokaryotic expression;
purification;
apoptosis detection;
sequence alignment
- From:
Acta Pharmaceutica Sinica
2017;52(6):904-910
- CountryChina
- Language:Chinese
-
Abstract:
Annexin is a protein of evolutionarily conserved polygene family that binds to cell membrane phosphatidylserine (PS). PS is closely related to many diseases with a potential as a new drug target. Annexin has a good value in drug discovery and new drug development. Annexin A4 is a member of the annexins family. Annexin A4 involves in a number of cellular functions, such as exocytosis and coagulation. These functions are related to binding of annexin to acidic phospholipids. However, the detail function(s) of annexin A4 has not been fully uncovered. Production of annexin A4 in large quantity is prerequisite for indepth investigation of the structure-function relationship of annexin A4. Human annexin A4 was originally purified from the natural resource at a low yield due to the complex procedure. In the present study, annexin A4 was expressed in a prokaryotic system with a high yield of soluble protein. The plasmid pET28a-annexin A4-EGFP was constructed for the expression. Recombinant annexin A4-EGFP was purified using two methods. Affinity chromatography approach gave a protein yield at purity of 80%. While, the membrane absorption method produced the protein with the purity over 90%. Flow cytometric analysis showed that the annexin A4-EGFP fusion protein could recognize and bind to the apoptotic cells with an affinity PS at 79.58±11.68 nmol·L-1, which is at the same order of magnitude as A5-EGFP. We successfully achieved the efficient expression of annexin A4-EGFP in prokaryotic system, and provided an easy and convenient method for purifying a large amount of annexin A4-EGFP with a high purity. This study has laid a solid foundation for our study of the function of annexin A4 in the future.
