TY - JOUR
T1 - A two-dimensional support for selective binding of polyhistidine-tagged proteins
T2 - Identification of a proliferating cell nuclear antigen point mutant with altered function in vitro
AU - Zaika, Alexander
AU - Mozzherin, Dmitry Ju
AU - Tan, Cheng Keat
AU - Downey, Kathleen M.
AU - Fisher, Paul A.
PY - 1999/3/15
Y1 - 1999/3/15
N2 - 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.
AB - 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.
KW - His-tag
KW - PCNA
KW - Template lesion bypass
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U2 - 10.1006/abio.1998.3074
DO - 10.1006/abio.1998.3074
M3 - Article
C2 - 10075808
AN - SCOPUS:0033559937
SN - 0003-2697
VL - 268
SP - 193
EP - 200
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -