Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation

Eileen M Lafer, Rui J Sousa, Alexander Rich

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Z-DNA affinity adsorbtion of an Escherichia coli lysate in the presence of excess B-DNA results in a 1000-fold enrichment for three proteins with apparent molecular weights, on SDS/polyacrylamide gel electrophoresis, of 50,000, 90,000 and 100,000. When retention of these proteins on resins constructed with Z-DNA (Br-poly(dG-dC) · poly(dG-dC)) was compared with retention on resins constructed with B-DNA or Br-B-DNA, it was found that approximately 100-fold more of the 50,000 Mr protein, 1000-fold more of the 90,000 Mr protein, and greater than 1000-fold more of the 100,000 Mr protein was retained on the Z-DNA resin. No difference inretention on the B-DNA versus brominated B-DNA resin was found, indicating that the increased retention on the Z-DNA resin was not due to bromination of the Z-DNA. This demonstration of Z-DNA-specific binding in vitro makes these proteins candidates for binding to Z-DNA in vivo. In an effort to determine the function of these proteins we have prepared monoclonal antibodies against each protein and isolated its respective gene. Western blot analysis of lysogens carrying these genes confirms their identity and shows that the complete coding region and promoter for each gene has been cloned.

Original languageEnglish (US)
Pages (from-to)511-516
Number of pages6
JournalJournal of Molecular Biology
Volume203
Issue number2
DOIs
StatePublished - Sep 20 1988
Externally publishedYes

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Z-Form DNA
DNA-Binding Proteins
B-Form DNA
Monoclonal Antibodies
Escherichia coli
Genes
Proteins
Halogenation
Genetic Promoter Regions
Protein Binding
Polyacrylamide Gel Electrophoresis
Molecular Weight
Western Blotting
DNA

ASJC Scopus subject areas

  • Virology

Cite this

Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation. / Lafer, Eileen M; Sousa, Rui J; Rich, Alexander.

In: Journal of Molecular Biology, Vol. 203, No. 2, 20.09.1988, p. 511-516.

Research output: Contribution to journalArticle

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