Wisén S, Jiang F, Bergman B, Mannervik B
Biomedical Center, Uppsala University, Uppsala, S-751 23, Sweden.
Protein Expr Purif. 1999 Dec;17(3):351-7. 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 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.
来自鱼腥藻Anabaena PCC 7120的转录因子NtcA在大肠杆菌中进行了异源表达。为了优化NtcA的表达，在编码该蛋白质的DNA的5'端引入了随机沉默突变。为了尽可能获得高产量的纯蛋白，采用了不同的表达策略以及纯化条件。在最佳表达条件下，NtcA的高水平表达克隆与GroEL-ES在37℃下共表达。在该蛋白质的N端添加了一个六组氨酸标签，以便在IMAC亲和柱上进行纯化。通过IMAC亲和色谱进行一步纯化后的表达，每升细菌培养物可产生30-40mg纯NtcA蛋白。凝胶迁移实验表明，重组NtcA在结合含有NtcA特异性位点的DNA序列方面具有活性。
