Production of recombinant human apoferritin heteromers.
We are interested in learning how iron is safely inserted and stored in ferritin. Recombinant DNA technology has considerable potential in determining the functional roles of the two ferritin subunits (H and L). In previous studies, we have observed that recombinant rat H ferritin was repressive to cell growth in both prokaryotic and eukaryotic expression systems (Guo et al., Biochem. Biophys. Res. Commun. 242, 39-45 (1998)). This results in the protein being expressed at very low levels. This problem was partially bypassed by the use of an inducible expression system, which utilizes T7 RNA polymerase dependent expression of the gene, induced by isopropyl beta-D-thiogalactopyranoside (IPTG). Simultaneously expressing the H and L ferritin genes in this system resulted in only a narrow range of ferritin heteromers, which predominantly consisted of the L subunit. Addition of rifampicin to cultures, 1 h following the induction of protein synthesis by IPTG, increased the production of the H subunit and thus increased the range of ferritin H:L subunit ratios. Simultaneous expression of the H and L ferritin genes in Escherichia coli grown in a deficient medium with minimal iron and with the addition of rifampicin resulted in the production of a range of recombinant human apoferritin heteromers that could be separated based on their subunit composition.
Grace
J E
JE
Biotechnology Center, Utah State University, Logan 84322, USA.
Van Eden
M E
ME
Aust
S D
SD
D001052:Apoferritins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D002570:Ceruloplasmin / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006801:Humans; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D012293:Rifampin / Q000494:pharmacology; D014162:Transfection
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D001052:Apoferritins; D002570:Ceruloplasmin; D012293:Rifampin