Molecular cloning and high-level expression of human polymerase β cDNA and comparison of the purified recombinant human and rat enzymes

Thomas A. Patterson, Wayne Little, Xinbo Cheng, Steven Widen, Amalendra Kumar, William A. Beard, Samuel H. Wilson

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

The cDNA encoding the human polymerase β from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase β cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase β mRNA in Escherichia coli and all yielded a high level of human polymerase β. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)100-110
Number of pages11
JournalProtein Expression and Purification
Volume18
Issue number1
DOIs
StatePublished - Jan 1 2000

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Molecular Cloning
Complementary DNA
Enzymes
HeLa Cells
Recombinant Proteins
Immunoglobulin G
Enzyme-Linked Immunosorbent Assay
Monoclonal Antibodies
Escherichia coli
Polymerase Chain Reaction
Messenger RNA

ASJC Scopus subject areas

  • Biotechnology

Cite this

Molecular cloning and high-level expression of human polymerase β cDNA and comparison of the purified recombinant human and rat enzymes. / Patterson, Thomas A.; Little, Wayne; Cheng, Xinbo; Widen, Steven; Kumar, Amalendra; Beard, William A.; Wilson, Samuel H.

In: Protein Expression and Purification, Vol. 18, No. 1, 01.01.2000, p. 100-110.

Research output: Contribution to journalArticle

Patterson, Thomas A. ; Little, Wayne ; Cheng, Xinbo ; Widen, Steven ; Kumar, Amalendra ; Beard, William A. ; Wilson, Samuel H. / Molecular cloning and high-level expression of human polymerase β cDNA and comparison of the purified recombinant human and rat enzymes. In: Protein Expression and Purification. 2000 ; Vol. 18, No. 1. pp. 100-110.
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