Expression and purification of the transcription factor NtcA from the cyanobacterium Anabaena PCC 7120

The transcription factor NtcA from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli. In order to optimize the exp ression of NtcA, random silent mutations were introduced at the 5' end of t he DNA encoding the protein. To get as high a yield of pure protein as poss ible, different strategies of expression as well as purification conditions were used. Under optimal expression conditions, a high-level expression cl one of NtcA was coexpressed with GroEL-ES at 37 degrees C, A hexahistidine tag was added to the N-terminus of the protein in order to allow purificati on on an IMAC affinity column. Expression followed by one purification step using IIMAC affinity chromatography gave a yield of 30-40 mg pure NtcA pro tein per liter of bacterial culture. Gel-shift experiments showed that the recombinant NtcA was active in binding a DNA sequence containing an NtcA-sp ecific site. (C) 1999 Academic Press.