High-level production of the metal-containing proteins in escherichia coli and their electrochemical properties

Abstract

To investigate the functions and properties of the metal-containing protein as an electron carrier, the expression of the recombinant protein is essential. For the expression of the fully active recombinant protein, the conversion of the expressed apoprotein into the holoprotein is the most important and difficult problem. Active recombinant mouse and Bacillus ferredoxins, iron-sulfur cluster proteins, have been obtained by overexpression in E. coli. A system for the production of the heme-containing protein as a pure recombinant holoprotein form was developed using the bovine cytochrome b5 tryptic fragment and E. coli bacterioferritin as heterologous and homologous heme-containing model proteins, respectively. This system was based on the slow synthesis of the recombinant apoprotein, which could maintain the balanced consumption of amino acids between protein synthesis and heme synthesis, so that the synthesized apoprotein could continue to act as a heme sink. This expression system provides a rapid and simple method obtaining large quantities of the active holo-form of the heme-containing proteins. Horse heart cytochrome c and recombinant cytochrome b5 have shown clear electrochemical responses on gold electrode modified with thiol compounds. In the case of ferredoxins, however, electron transfer between modified gold electrode and the protein did not occurred. In addition, horse spleen ferritin, an iron storage protein, did not have influence upon the electrochemistry of iron on a gold surface.