Production of Human RAP30 and RAP74 in Bacterial Cells

Abstract

RAP30 and RAP74 are subunits of RAP30/74 (TFIIF), a general initiation and elongation factor for transcription by RNA polymerase II. Complementary DNA (cDNA) clones have previously been reported encoding human RAP30 and RAP74. Here we report expression of these cDNAs using a T7 RNA polymerase system in Escherichia coli. Production of human RAP30 was very efficient using the expression vector pET1 1d. RAP30 accumulated within inclusion bodies and was solubilized using guanidine hydrochloride. After removal of the denaturant, RAP30 was soluble and active in accurate transcription. Approximately 44 mg of highly purified and soluble RAP30 was obtained from a 1-liter culture of cells. Production of RAP74 was more problematic, because a mixture of full length RAP74 and RAP74 fragments was produced in E. coli. Most RAP74 fragments were shortened by deletion of the COOH-terminus of the protein and probably resulted from premature translation termination. RAP74 was most successfully produced using a pET23d construct, in which the RAP74 peptide was fused to a short polyhistidine stretch at its COOH-terminus. Addition of the polyhistidine sequence allowed purification using a Ni 2+ affinity resin. Full length RAP74 carrying this polyhistidine extension was purified in a single step by Ni 2+ affinity chromatography in 4 M urea; the yield of RAP74 was approximately 3 mg from a 1-liter culture of cells. RAP 74 derivatized with a polyhistidine stretch at its NH 2-terminus, on the other hand, remained contaminated with RAP74 fragments after Ni 2+ affinity chromatography. These fragments were dissociated from RAP74 in 4 M urea and separated by Mono Q and Mono S chromatography; approximately 800 μg pure, intact, and active RAP74 was obtained from a 1-liter culture of cells using this procedure.