Metal-Binding Activity of the Soluble Recombinant Pig Metallothionein 1A Expressed in Escherichia coli

Abstract

Full-length cDNA for the pig metallothionein 1A (pMT1A) gene was synthesized based on the pig MT1A gene sequence in Genbank and cloned into the pMD18-T vector. After sequence analysis and structure prediction, the pMT1A gene was cloned into vector pET-32a (+) containing a His-tag. The recombinant pMT1A (rpMT1A) was expressed in a soluble form using Escherichia coli Rosetta™ (DE3) plysS cells. Western blotting showed that the purified rpMT1A protein bound an anti-His-tag monoclonal antibody. Further investigation revealed that the rpMT1A protein showed high metal-binding activity with the divalent metal ions copper (Cu²⁺), zinc (Zn²⁺), and cadmium (Cd²⁺).

A Polyclonal Antibody Against Recombinant Bovine Haptoglobin Expressed inEscherichia coli

“…At present, many purification methods for recombinant proteins generated in prokaryotic expression systems have been reported and include affinity chromatography and ion exchange chromatography, as well as purification methods based on tag proteins, gel-cutting purification, and so on. (21)(22)(23)(24) Of these protein purification methods, gel-cutting purification is a simple, time-saving, and much less expensive approach to the preparation of high-purity recombinant proteins in vitro. In our study, the cutting-gel purified proteins were dialyzed using the D-TubeÔ Dialyzer (EMD Millipore, Darmstadt, Germany) to remove the SDS.…”