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

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.