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

Whatman 3MM paper was chemically modified to generate nickel-charged iminod iacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escheri chia coil expression plasmids coding for either unmodified proliferating ce ll nuclear antigen (PCNA) or PCNA containing a genetically engineered polyh istidine tract (his-tag) located at its NH, 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 acti ve in an in situ DNA synthesis assay with exogenous template-primer and pur ified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promote s increased DNA synthesis by pol delta beyond a model abasic template site, In addition, metal-charged IDA paper promises to be generally useful for f unctional screening of cells expressing cloned proteins. (C) 1999 Academic Press.