Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli

Journal Article · Tue Oct 29 00:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00107a009· OSTI ID:5687435

Cherney, B W; Bhatia, K; Smulson, M ^[1]; Chaudhry, B ^[2]; Butt, T R ^[3]

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1. Georgetown Univ., Washington, DC (United States)
2. Punjab Univ., Lahore (Pakistan)
3. Smith Kline Beechman Pharmaceuticals, King of Prussia, PA (United States)

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. 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. The authors further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.

• OSTI ID: 5687435
• Journal Information: Biochemistry; (United States), Vol. 30:43; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
POLYMERASES
AUTORADIOGRAPHY
RECOMBINANT DNA
GENE RECOMBINATION
BIOCHEMICAL REACTION KINETICS
ELECTROPHORESIS
ENZYME ACTIVITY
ESCHERICHIA COLI
GENE REPRESSORS
HELA CELLS
MUTAGENESIS
PHOSPHORUS 32
PLASMIDS
POST-TRANSLATION MODIFICATION
STRAND BREAKS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DNA
ENZYMES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOPROTEINS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques