Purification of recombinant human transcription factor IIB by immunoaffinity chromatography.
The human RNA polymerase II transcription factor IIB (TFIIB) was purified from a bacterial expression system by immunoaffinity chromatography. Mouse monoclonal antibodies (MAbs) were prepared that react with TFIIB. A modified enzyme-linked immunosorbent assay was used to screen for MAbs that release the antigen in the presence of a low molecular weight polyhydroxylated compound and a nonchaotropic salt (polyol-responsive MAbs). One polyol-responsive MAb (designated IIB8) was purified by chromatography on protein A and conjugated to cyanogen bromide-activated Sepharose 4B. Escherichia coli strain BL21 (DE3) containing the pLysS plasmid was transformed with the human TFIIB gene contained in the pET11a vector (phIIB). After induction with IPTG, the cells were harvested and lysed. The lysate was treated with 0.5% polyethyleneimine and centrifuged. The supernatant fluid was applied to the IIB8-Sepharose. After extensive washing, the TFIIB was eluted with Tris-EDTA buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. The purified TFIIB was active when added back to TFIIB-depleted HeLa nuclear extract and when used in the IgH minimal promoter system. This method will be useful for the rapid purification of TFIIB mutants and for the purification of large amounts of highly purified TFIIB for biochemical studies. In addition, this procedure establishes the general applicability of the use of polyol-responsive MAbs for the rapid, gentle purification of labile proteins.
Thompson
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NE
McArdle Laboratory for Cancer Research, University of Wisconsin at Madison 53706.
Burgess
R R
RR
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors