J Integr Plant Biol. ›› 2003, Vol. 45 ›› Issue (1): 98-101.
• Research Articles •
ZHOU Jie,HAO Fu-Ying,SHI Ding-Ji,YU Mei-Min,RU Bing-Gen
To produce mouse metallothionein_Ⅰ (mMT_Ⅰ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia coli_cyanobacterium shuttle fusion expression vector, pKG_MT, was constructed. Via this vector, mMT_Ⅰ cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione_S_transferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS_polyacrylamid gel electrophoresis (SDS_PAGE) showed that the fusion protein GST_MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio_β_D_galactoside (IPTG). Glutatione_S_transferase metallothionein (GST_MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT_Ⅰ was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G_50. SDS_PAGE demonstrated that the purified mMT_Ⅰ was the desired protein. The result of ELISA for the purified mMT_Ⅰ showed that the recovery of mMT_Ⅰ from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal_binding activity of the purified mMT_Ⅰ was almost the same as that of wild type MT.
ZHOU Jie,HAO Fu-Ying,SHI Ding-Ji,YU Mei-Min,RU Bing-Gen. Expression of Mouse MT-Ⅰ as a Fusion Protein in Anabaena sp. PCC 7120[J]. J Integr Plant Biol., 2003, 45(1): 98-101.
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