Recombinant ferritin: modulation of subunit stoichiometry in bacterial expression systems.
We describe a strategy for the creation of recombinant ferritin heteropolymers which mimic the natural heterogeneity of this protein. This method entailed the co-expression of cDNA for both ferritin H and ferritin L subunits in a single bacterium using either a bicistronic vector, in which both cDNAs were expressed from the vector, or a dual vector expression strategy, in which each subunit was expressed from a separate compatible plasmid in a single bacterial host. Electron microscopy and sucrose density gradient centrifugation demonstrated that ferritin assembled spontaneously in such bacteria to form catalytically active proteins of the expected size and shape. Isoelectric focusing revealed that protein isolated from any of these bacteria exhibited a restricted heterogeneity in subunit composition. Such multi-subunit recombinant ferritins spontaneously assembled in bacteria may be useful in further studies of ferritin assembly and function. Our results further suggest that varying expression levels is a simple way to alter levels of individual components within a multi-subunit recombinant protein, and that this approach may be of general utility in assessing the contribution of individual components to the function of multi-subunit proteins or protein complexes.
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Department of Cancer Biology, Bowman Gray School of Medicine and the Comprehensive Cancer Center of Wake Forest University, Winston-Salem, NC 27157, USA.
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D000818:Animals; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007501:Iron / Q000032:analysis; D007525:Isoelectric Focusing; D051379:Mice; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron