Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody.
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.
Thompson
N E
NE
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Aronson
D B
DB
Burgess
R R
RR
D000645:Ammonium Sulfate; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012319:RNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000201:enzymology; D014908:Triticum / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D005026:Ethylene Glycols; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D012319:RNA Polymerase II; D019855:Ethylene Glycol; D000645:Ammonium Sulfate