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.

Zaika A, Mozzherin DJ, Tan CK, Downey KM, Fisher PA
Anal Biochem. 1999 268 (2): 193-200

PMID: 10075808 · DOI:10.1006/abio.1998.3074

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-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 delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta 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.

Copyright 1999 Academic Press.

MeSH Terms (17)

Animals Base Sequence Binding Sites Cattle DNA DNA Polymerase III DNA Primers Drosophila Escherichia coli Histidine In Vitro Techniques Paper Point Mutation Proliferating Cell Nuclear Antigen Recombinant Proteins Thymus Gland Transformation, Genetic

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