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 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 degrees 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.
Wisén
S
S
Biomedical Center, Uppsala University, Uppsala, S-751 23, Sweden.
Jiang
F
F
Bergman
B
B
Mannervik
B
B
D000595:Amino Acid Sequence; D017033:Anabaena / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D018835:Chaperonin 10 / Q000096:biosynthesis; D018834:Chaperonin 60 / Q000096:biosynthesis; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C075617:ntcA protein, Synechococcus