Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli.
The cDNA of human poly(ADP-ribose) polymerase (pADPRP), encoding the entire protein, was subcloned into the Escherichia coli expression plasmid pYUb. In this expression system, the carboxyl terminus of ubiquitin is fused to the amino terminus of a target protein, in this case pADPRP, stabilizing the accumulation of the cloned gene product. Following induction of the transformed cells, the sonicated extract contained a unique protein immunoreactive with both pADPRP and ubiquitin antibodies and corresponding to the predicted mobility of the fusion protein in SDS-PAGE. Fusion of ubiquitin to pADPRP increased the yield of pADPRP approximately 10-fold compared to that of the unfused enzyme. The resulting recombinant fusion protein had catalytic properties which were nearly identical to those of native pADPRP obtained from mammalian tissues. These properties included specific activity, Km for NAD, response to DNA strand breaks, response to Mg2+, inhibition by 3-aminobenzamide, and activity in activity gel analysis. An initial analysis by deletion mutagenesis of pADPRP's functional domains revealed that deletions in the NAD binding domain eliminated all activity; however, partial polymerase activity resulted from deletion in the DNA binding or automodification domains. The activities were not enhanced by breaks in DNA. We further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, D.C. 20007.
B W
BW
Cherney
B
B
Chaudhry
K
K
Bhatia
T R
TR
Butt
M
M
Smulson
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D016296:Mutagenesis; D010957:Plasmids; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D011065:Poly(ADP-ribose) Polymerases