Synonymous mutations in the second and the fourth codons stimulate the expression of human cardiac troponin I gene in E.coli
- VernacularTitle:人心肌肌钙蛋白I第2和第4个密码子同义突变对其在大肠埃希菌中表达的影响
- Author:
Hongmei LIU
;
Desheng LUO
;
Shen QU
- Publication Type:Journal Article
- Keywords:
troponin I;
point mutation;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(09):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To induce the site-directed mutation of human cardiac troponin I (cTnI) gene, express the mutant in E. coli, and to study the effects of the mutation on the prokaryotic expression of cTnI. Methods The cDNA encoding cTnI was cloned with RT-PCR from the total RNA extracted from human myocardium tissues. A pair of primers was designed and, after the mutations were induced at the second and the fourth codons, inserted into prokaryotic vector pET-28c (+) and transform the recombinant to BL21 (DE3) bacteria. After purified with Ni-NTA resin, the histidine-tagged fusion protein expressed by IPTG-induced was identified by Western blotting and the expression yield of cTnI protein was investigated. Results The expression of the recombinant carrying processed cTnI cDNA was stronger than that in control group. Conclusion cDNA encoding cTnI was successfully cloned. The recombinant with mutations can be more efficient expressed in E. coli. The cTnI protein can be purified to near homogeneity.
