Abstract
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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Base Sequence
-
Chromatography, Gel
-
DNA-Directed RNA Polymerases / metabolism
-
Electrophoresis, Polyacrylamide Gel
-
Escherichia coli / genetics*
-
Gene Expression*
-
Gene Transfer Techniques
-
Glutathione Transferase / genetics
-
Molecular Sequence Data
-
Molecular Weight
-
Plasmids
-
RNA, Transfer, Arg / genetics
-
Recombinant Fusion Proteins / isolation & purification
-
Saccharomyces cerevisiae / chemistry
-
Saccharomyces cerevisiae / genetics*
-
Transcription Factor TFIIIA
-
Transcription Factors / genetics*
-
Transcription Factors / isolation & purification
-
Transcription Factors / metabolism
-
Viral Proteins
Substances
-
RNA, Transfer, Arg
-
Recombinant Fusion Proteins
-
Transcription Factor TFIIIA
-
Transcription Factors
-
Viral Proteins
-
Glutathione Transferase
-
bacteriophage T7 RNA polymerase
-
DNA-Directed RNA Polymerases