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.[1]References
- Immunoaffinity purification of the RAP30 subunit of human transcription factor IIF. Thompson, N.E., Burgess, R.R. Protein Expr. Purif. (1999) [Pubmed]
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