A two-dimensional support for selective binding of polyhistidine-tagged proteins: Identification of a proliferating cell nuclear antigen point mutant with altered function in vitro

Alexander Zaika, Dmitry Ju Mozzherin, Cheng Keat Tan, Kathleen M. Downey, Paul A. Fisher

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni26+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase δ (pol δ). Ni2+- IDA paper was used to identify a PCNA- point mutant that, relative to wild- type PCNA, promotes increased DNA synthesis by pol δ beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Original languageEnglish (US)
Pages (from-to)193-200
Number of pages8
JournalAnalytical Biochemistry
Volume268
Issue number2
DOIs
StatePublished - Mar 15 1999
Externally publishedYes

Keywords

  • His-tag
  • PCNA
  • Template lesion bypass

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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