Characterization and site-specific mutagenesis of the calcium-binding protein oncomodulin produced by recombinant bacteria.
A bacterial expression system for the parvalbumin-like calcium-binding protein oncomodulin has been constructed. This system can yield 50-fold more oncomodulin than the richest known mammalian source, the rat Morris hepatoma 5123. The bacterially produced protein folded correctly as monitored by UV, fluorescence, and 1H nuclear magnetic resonance spectroscopy and is immunologically identical to rat hepatoma oncomodulin. A calcium-specific conformational change is observed in the bacterial oncomodulin similar to that of the hepatoma protein. A modification of the putative calcium-specific CD loop by site-directed mutagenesis, which changed Asp-59 to Glu-59, eliminates calcium-specific effects. In contrast to the native molecule, the mutant Glu-59 now exhibits a magnesium-induced conformational change when monitored by UV difference or fluorescence excitation spectroscopy. The availability of large amounts of bacterially produced oncomodulin combined with the ability to modify at will the metal-binding ligands should now allow dissection of the unusual pairing in oncomodulin of one calcium-specific calmodulin-like site with one calcium/magnesium parvalbumin-like site.
MacManus
J P
JP
Division of Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Hutnik
C M
CM
Sykes
B D
BD
Szabo
A G
AG
Williams
T C
TC
Banville
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D009363:Neoplasm Proteins; D010957:Plasmids; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002135:Calcium-Binding Proteins; D004274:DNA, Recombinant; D009363:Neoplasm Proteins; C034733:oncomodulin; D008274:Magnesium; D002118:Calcium