Gene cloning, expression, and purification of recombinant human heat shock protein 70D
- VernacularTitle:人热休克蛋白70D的基因克隆、表达及蛋白纯化
- Author:
Weihong CHANG
;
- Publication Type:Journal Article
- Keywords:
heat shock protein 70;
gene cloning;
recombinant expression;
purification
- From:
Academic Journal of Second Military Medical University
2000;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To study the cloning, recombinant expression and purification of human HSP70D. Methods: The full length code region of HSP70D was amplified by RT PCR from HeLa cells. Then the PCR product was inserted into pET 24a(+), by NdeⅠ/XhoⅠ double digestion. The recombinant expression vector pET24a HSP70D was transformed into E. coli strain BL21. Positive clones were determined by NdeⅠ/XhoⅠ digestion and sequence analysis, which was used in the expression and purification of HSP70D protein. The engineered bacteria were collected after fermentation. The recombinant HSP70D was purified by using ethanol precipitation, anion exchanger, hydrophobicity interaction chromatography, and finally high resolution gel filtration chromatography. Results: The expression level of HSP70D was over 20% of total protein in the engineered bacteria. After purification, electrophoretically pure (more than 95%) rhHSP70D was obtained. Conclusion: This study setup a satisfied down stream process which can be used for production of large quantity of recombinant HSP70D.
