[Cloning, expression and purification of rabbit metallothionein-I gene in Escherichia coli].
The cDNA encoding the rabbit metallothionein-I was amplified by RT-PCR from the rabbit liver induced by cadmium and cloned into prokaryotic fusion expression vector pQE40. Then it was transformed into Escherichia coli M15. Positive expression clones were detected by colony blotting. Target protein solubility was determined by Western blotting analysis. The optimal induction condition of the level of protein expression with IPTG induction was established by SDS-PAGE electrophoresis and ImageMaster VDS software analysis. The fusion protein can be purified from lysates with Ni-NTA agarose. We found that the fusion protein with apparent molecular weight 32 KD existed in two ways: soluble and insoluble in Escherichia coli. After 1 mM IPTG induction, the level of expression of the fusion protein increased with the prolongation of induction time and reached a peak in 9 h by ImageMaster VDS software analysis, accounting for 57.4% of all the insoluble protein. The purified fusion protein was obtained by Ni-NTA affinity chromatography. This fusion protein can be used in further studies on the preparation of MT-I protein and development of protein product.
Sun
Ying
Y
Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, China.
He
Zhimin
Z
Yang
Fang
F
Chen
Zhuchu
Z
Yan
Bin
B
Huang
Hongke
H
Li
Tingbao
T
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014169:Transformation, Bacterial
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D008668:Metallothionein