Immunoaffinity purification of the RAP30 subunit of human transcription factor IIF.
RAP30, an RNA polymerase-associated protein (RAP) of approximately 30 kDa, is a component of the eukaryotic general transcription factor IIF (TFIIF). We have isolated a monoclonal antibody (MAb) that can be used to purify RAP30 under nondenaturing conditions. This MAb (designated 1RAP1) is a unique type of MAb that we have designated "polyol-responsive MAb." Polyol-responsive MAbs are high-affinity antibodies that release antigen in a buffer containing a low-molecular-weight polyhydroxylated compound (polyol) and a nonchaotropic salt. RAP30, contained on pET11d, was expressed in Escherichia coli by culturing and inducing protein expression at 26 degrees C. Under these conditions, approximately 50% of the RAP30 remains soluble. Inclusion bodies were removed from the cell lysate by centrifugation, the supernatant was treated with polyethyleneimine at 0.5 M NaCl to remove nucleic acids, and the soluble protein was applied directly to MAb-conjugated Sepharose. After extensive washing, RAP30 was eluted with buffer containing 0. 75 M ammonium sulfate and 40% propylene glycol. RAP30 produced by this procedure stimulates transcription from a minimal promoter. This is a rapid method for purifying unmodified RAP30 without renaturing the protein from inclusion bodies.
Thompson
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NE
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Burgess
R R
RR
D000665:Ampholyte Mixtures / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D007074:Immunoglobulin G; D002479:Inclusion Bodies / Q000378:metabolism; D011108:Polymers / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification; D012685:Sepharose / Q000378:metabolism; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification*; D019473:Transcription Factors, TFII; D014158:Transcription, Genetic
D000665:Ampholyte Mixtures; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011108:Polymers; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C024617:polyol; D012685:Sepharose; D017346:Protein Serine-Threonine Kinases; C056604:transcription factor TFIIF