The carboxyl-terminal domain of human poly(ADP-ribose) polymerase. Overproduction in Escherichia coli, large scale purification, and characterization

• Published in: The Journal of biological chemistry Vol. 268; no. 18; pp. 13454 - 13461
• Main Authors: SIMONIN, F, HÖFFERER, L, PANZETER, P. L, MULLER, S, DE MURCIA, G, ALTHAUS, F. R
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.06.1993
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Catalysis; Chromatography, Ion Exchange; Cloning, Molecular; DNA - metabolism; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Humans; Molecular Sequence Data; NAD - metabolism; NAD+ Nucleosidase - metabolism; Poly(ADP-ribose) Polymerases - biosynthesis; Poly(ADP-ribose) Polymerases - genetics; Poly(ADP-ribose) Polymerases - isolation & purification; Poly(ADP-ribose) Polymerases - metabolism; Transferases; Human; Purification; Enzymatic activity; C terminal peptide; Enzyme; NAD+ ADP-ribosyltransferase; DNA; Recombinant protein; Histone
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)38671-5

The cDNA encoding the carboxyl-terminal 40-kDa domain of human poly(ADP-ribose) polymerase was inserted into an expression vector. The recombinant protein was overproduced in Escherichia coli, and purified to homogeneity. The 40-kDa domain had the same affinity (Km) for NAD+ as the full-length enzyme, expressed abortive NAD+ glycohydrolase activity, catalyzed the initiation, elongation, and branching of ADP-ribose polymers, but exhibited no DNA dependence. Its specific activity was approximately 500-fold lower than that of the whole enzyme activated by DNA strand breaks. Surprisingly, the carboxyl-terminal 40-kDa domain exhibited the processive mode of polymer attachment typical of full-length poly(ADP-ribose) polymerase and was able to modify histones H1 and H2B. Finally, the polymer sizes formed by the 40-kDa domain were influenced by histone H1.