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J. Biol. Chem., Vol. 265, Issue 12, 7069-7077, Apr, 1990
NE Thompson, DB Aronson and RR Burgess
Active eukaryotic RNA polymerase II (RNAP II) was purified by
immunoaffinity chromatography, using a monoclonal antibody (mAb) that
reacts with the highly conserved heptapeptide repeat of the largest
subunit. This mAb (designated SWG16) was conjugated to CNBr-activated
Sepharose and used to purify RNAP II from wheat germ and calf thymus. The
subunit composition of the immunoaffinity-purified enzyme was essentially
the same as RNAP II purified by conventional chromatography except that it
contained only the form with the unproteolyzed largest subunit. Active
enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with
combinations of low molecular weight polyols and nonchaotropic salts. The
superior eluting procedure used combinations of ethylene glycol (30-40%)
and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with
a synthetic peptide containing four repeats of the heptapeptide; however,
the peptide was not as effective as the polyol and salt combinations for
eluting the enzyme. This mAb should be useful for purifying RNAP II from
many eukaryotic species. Because the elution of enzyme from the
immunoadsorbent seems to be dependent upon the presence of a polyol, this
antibody is referred to as a "polyol-responsive mAb." A procedure that
helps to identify a polyol-responsive mAb and to optimize the eluting
conditions is described. Polyol-responsive mAbs might have broad
applicability to the purification of many labile enzymes by immunoaffinity
chromatography.
Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
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