RECOMBINANT FERRITIN - MODULATION OF SUBUNIT STOICHIOMETRY IN BACTERIAL EXPRESSION SYSTEMS

We describe a strategy for the creation of recombinant ferritin hetero polymers which mimic the natural heterogeneity of this protein. This m ethod entailed the co-expression of cDNA for both ferritin H and ferri tin 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 separ ate compatible plasmid in a single bacterial host, Electron microscopy and sucrose density gradient centrifugation demonstrated that ferriti n assembled spontaneously in such bacteria to form catalytically activ e proteins of the expected size and shape. Isoelectric focusing reveal ed that protein isolated from any of these bacteria exhibited a restri cted heterogeneity in subunit composition, Such multi-subunit recombin ant ferritins spontaneously assembled in bacteria may be useful in fur ther studies of ferritin assembly and function, Our results further su ggest that varying expression levels is a simple way to alter levels o f individual components within a multi-subunit recombinant protein, an d that this approach may be of general utility in assessing the contri bution of individual components to the function of multi-subunit prote ins or protein complexes.