Expression in Escherichia coli: purification and properties of the yeast general transcription factor TFIID.
A T7 RNA polymerase expression system has been used for the efficient expression of the yeast RNA polymerase general transcription factor TFIID (TFIIDY), the TATA-box factor (previously called BTF1) in Escherichia coli. Expression of the gene was performed at 25 degrees C instead of 37 degrees C to increase the total amount of soluble TFIIDY. Soluble TFIIDY was purified in three chromatographic steps and was eluted from the final column, a heparin-5PW HPLC column, in two peaks at 0.38 M (peak I) and 0.42 M (peak II) KCl in which this protein was 52% and greater than 95% pure, respectively. The protein in both peaks was active in an in vitro transcription assay. However, while TFIIDY from peak II was essentially indistinguishable from the material isolated from yeast, the protein of peak I differed in a number of biochemical characteristics, having a lower specific activity in an in vitro transcription assay and displaying an altered pattern of bands in a DNA band shift assay. Despite these differences, the proteins in both peaks have identical molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable N-terminal amino acid sequences, and apparently exist as monomers under the conditions used for the heparin-5PW chromatography.
Burton
N
N
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Faculté de Médecine, Strasbourg, France.
Cavallini
B
B
Kanno
M
M
Moncollin
V
V
Egly
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D002845:Chromatography / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors