title: Fast purification of the Apo form and of a non-binding heme mutant of recombinant sperm whale myoglobin.
abstract: As molecular biology has developed it has become possible to abundantly produce heterologous proteins in bacteria and to design serial amino acid substitutions for the generation of modified proteins, an approach also known as protein engineering. Sperm whale myoglobin, a protein of broad interest, has been cloned for several years now and a large collection of mutants has been produced. The presence of heme stabilizes the protein, which is recovered soluble from the bacterial pellet, and most purification protocols take advantage of this property for myoglobin purification directly from the pellet. However, recovery from the column resin is poor with these methods making them expensive and the procedure for removing heme is laborious and drastic when the apo form of Mb is required. In the case of proteins with severe mutations, which bind heme weakly or do not bind it at all, such methods cannot be employed without massive loss of productivity. Here, we describe a modified method, which is both low cost and rapid, for the purification of the soluble apo form of Mb from Escherichia coli inclusion bodies. Biophysical characterization of the protein after purification shows that the purified apoMb retains its native conformation and is soluble. This modified method is also used for the purification of a non-heme-binding apoMb mutant, demonstrating its efficiency when dealing with drastic mutations.
authors.affiliation: Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, P.O. Box 6192, Campinas SP 13084-971, Brazil.
authors.forename: Euripedes A
authors.initials: EA
authors.lastname: Ribeiro
authors.forename: Wiliam C B
authors.initials: WC
authors.lastname: Regis
authors.forename: Ljubica
authors.initials: L
authors.lastname: Tasic
authors.forename: Carlos H I
authors.initials: CH
authors.lastname: Ramos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D014907:Whales
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D021122:Protein Subunits; D011994:Recombinant Proteins; D006418:Heme