High-level production of heme-containing holoproteins in Escherichia coli.
The expression of recombinant protein is essential for the investigation of the functions and properties of heme-containing protein as an electron carrier. For the expression of fully active recombinant protein, conversion of the expressed apoprotein into holoprotein is the most important and difficult problem. In this study, a system was developed for the production of heme-containing protein in a pure, recombinant holoprotein form, using the bovine cytochrome b5 tryptic fragment and Escherichia coli bacterioferritin as heterologous and homologous heme-containing model proteins, respectively. This system is based on the slow synthesis of recombinant apoprotein, which can maintain the balanced consumption of amino acids between protein synthesis and heme synthesis, so that the synthesized apoprotein continues to act as a heme sink. From a 1-1 culture, 15 mg of cytochrome b5 and 40 mg of bacterioferritin were purified as pure holoprotein forms. Our expression system provides a rapid and simple method for obtaining large quantities of the active holo-form of heme-containing proteins.
Jung
Y
Y
Department of Chemistry, School of Molecular Science BK21, Korea Advanced Institute of Science and Technology, Taejon.
Kwak
J
J
Lee
Y
Y
D000818:Animals; D001426:Bacterial Proteins; D002417:Cattle; D003573:Cytochrome b Group / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D011994:Recombinant Proteins; D005293:Ferritins; C018032:bacterioferritin; D015786:Cytochromes b5