Expression and Purification of the Transcription Factor NtcA from the Cyanobacterium Anabaena PCC 7120

• Published in: Protein expression and purification 1999-12, Vol.17 (3), p.351-357
• Main Authors: Wisén, Susanne, Jiang, Fanyi, Bergman, Birgitta, Mannervik, Bengt
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anabaena - metabolism; Bacterial Proteins; Base Sequence; Chaperonin 10 - biosynthesis; Chaperonin 60 - biosynthesis; Chromatography, Affinity; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Escherichia coli - metabolism; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Transcription Factors - biosynthesis; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1999.1131

The transcription factor NtcA from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli. In order to optimize the expression of NtcA, random silent mutations were introduced at the 5′ end of the DNA encoding the protein. To get as high a yield of pure protein as possible, different strategies of expression as well as purification conditions were used. Under optimal expression conditions, a high-level expression clone of NtcA was coexpressed with GroEL-ES at 37°C. A hexahistidine tag was added to the N-terminus of the protein in order to allow purification on an IMAC affinity column. Expression followed by one purification step using IMAC affinity chromatography gave a yield of 30–40 mg pure NtcA protein per liter of bacterial culture. Gel-shift experiments showed that the recombinant NtcA was active in binding a DNA sequence containing an NtcA-specific site.