Expression, purification of recombinant human cryptochrome I and its application in preparation of protective agent for radiotherapy.
- Author:
Chen YAO
;
Chunjie SHENG
;
Dong LIU
;
Shijuan GAO
;
Wei JIANG
;
Hongyan YU
;
Jiandong LI
;
Huiming CHEN
;
Jiaoxiang WU
;
Changchuan PAN
;
Shuai CHEN
;
Wenlin HUANG
- Publication Type:Journal Article
- MeSH:
Cryptochromes;
biosynthesis;
Escherichia coli;
Humans;
Plasmids;
Radiation-Protective Agents;
Recombinant Proteins;
biosynthesis
- From:
Chinese Journal of Biotechnology
2015;31(1):135-146
- CountryChina
- Language:Chinese
-
Abstract:
Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.
