Expression of Human Dna Polymerase β in Escherichia Coli and Characterization of the Recombinant Enzyme

John Abbotts, Dibyendu N. SenGupta, Barbara Zmudzka, Steven Widen, Vicente Notario, Samuel H. Wilson

Research output: Contribution to journalArticle

116 Citations (Scopus)

Abstract

The coding region of a human 0-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1–2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural 0-polymerases. The purified enzyme was free of nuclease activity. We studied detailed catalytic properties of the recombinant 0-polymerase using defined template-primer systems. The results indicate that this 0-polymerase is essentially identical with natural 0-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N3-methyl-dT or O6-methyl-dG.

Original languageEnglish (US)
Pages (from-to)901-909
Number of pages9
JournalBiochemistry
Volume27
Issue number3
DOIs
StatePublished - Feb 1 1988
Externally publishedYes

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Escherichia coli
Enzymes
Proteins
Complementary DNA
DNA-Directed DNA Polymerase
Complex Mixtures
Recombinant Proteins
Plasmids
Tissue
Amino Acids
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression of Human Dna Polymerase β in Escherichia Coli and Characterization of the Recombinant Enzyme. / Abbotts, John; SenGupta, Dibyendu N.; Zmudzka, Barbara; Widen, Steven; Notario, Vicente; Wilson, Samuel H.

In: Biochemistry, Vol. 27, No. 3, 01.02.1988, p. 901-909.

Research output: Contribution to journalArticle

Abbotts, John ; SenGupta, Dibyendu N. ; Zmudzka, Barbara ; Widen, Steven ; Notario, Vicente ; Wilson, Samuel H. / Expression of Human Dna Polymerase β in Escherichia Coli and Characterization of the Recombinant Enzyme. In: Biochemistry. 1988 ; Vol. 27, No. 3. pp. 901-909.
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