Single amino acid substitution enhances bacterial expression of PARP-4D214A.
Poly(ADP-ribose) polymerase-1 (PARP-1) is the canonical member of the PARP family of enzymes and modulates many crucial nuclear functions. PARP-1 is involved in apoptosis and is the substrate of caspase-3, a protease that cleaves PARP-1 at the conserved sequence 211DEVD214. To generate a caspase-3-uncleavable PARP-1, we introduced an amino acid substitution D214-->A214 at the site of cleavage. We observed that following over-expression in bacteria, the mutant protein HIS-PARP-1D214A was expressed several-fold more than a unmutated copy, HIS-PARP-1. The specific activity of HIS-PARP-1 enzyme in total bacterial extracts was 6.94 U/mg and 4.61 U/mg for HIS-PARP-1D214A. This approach should provide new avenues for crystallographic study of PARP-1 as well as new information for drug design targeting PARP-1.
Gagnon
Steve N
SN
Pediatrics Research Unit, CHUL Research Center of CHUQ and Laval University, Sainte-Foy, Quebec, Canada.
Desnoyers
Serge
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017354:Point Mutation; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013234:Stem Cells
D000596:Amino Acids; D018076:DNA, Complementary; D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases