High level expression in E. coli and purification of yeast transcription factor IIIA.
Saccharomyces cerevisiae transcription factor IIIA, a sequence-specific DNA binding protein that is required for transcription of 5S rRNA genes by RNA polymerase III, has been expressed in Escherichia coli in a full length, native form. High level expression was achieved through the combined use of a T7 RNA polymerase expression system and of a multicopy plasmid carrying an E. coli gene, argU, which codes for a minor Arg(AGA/AGG) tRNA species. Recombinant yeast transcription factor IIIA was purified to 95% homogeneity, at a final yield of 8 mg/liter of bacterial culture, by three chromatographic steps, and it was shown to be at least 55% active by quantitative in vitro transcription assays.
Ottonello
S
S
Institute of Biochemical Sciences, University of Parma, Italy.
Ballabeni
A
A
Soncini
C
C
Dieci
G
G
D001483:Base Sequence; D002850:Chromatography, Gel; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D012347:RNA, Transfer, Arg / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D012347:RNA, Transfer, Arg; D011993:Recombinant Fusion Proteins; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D014764:Viral Proteins; D005982:Glutathione Transferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases