Purification Strategies and Considerations in Overproduction, Isolation and Reconstitution of Labile Metalloproteins

摘要

Many proteins, even today, are unexpectedly found to purify with or require reconstitution of a metalloprotein cofactor in order to perform their function. These factors include metal ions themselves (Fe2+, Cu2+, Zn2+, Mg2+, Mn2+ etc.) bound by an arrangement of amino acid ligands from a protein. Additional cofactors range in complexity from ubiquitous and essential iron-sulfur clusters and tetrapyrrole derivatives (heme, chlorophylls, chlorins) to more complex and much less frequent molybdo(tungsto)pterin, hydrogenase and nitrogenase cofactors, the latter being found in a single enzyme, nitrogenase. Overproduction and specifically heterologous expression of proteins containing any of these metalloprotein cofactors can be complicated by the fact that even simple cofactors such as iron sulfur (FeS) clusters, require a dedicated multi-protein systems for assembly and insertion into apoproteins.