Expression of Mouse MT-Ⅰ as a Fusion Protein in Anabaena sp. PCC 7120

摘要

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-trans-ferase ( 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.