The Construction of T7 Promoter-based His(6)-tagging Vectors and the Single-step Purification of the Expression products.
T7 promoter-based fusion expression vectors have been constructed that directed the synthesis of heterologous proteins in E. coli as fusions with a stretch of six consecutive histidine residues His(6) at N Terminus. The vectors were also featured with strong T7 promoter, terminator, translational start, multiple cloning sites with seven unique restriction sites in all three reading frames and the f1 phage origin which allows the packaging of single-stranded plasmid, mutagenesis and DNA sequencing without subcloning steps. In most cases, expressed fusion proteins are soluble. The His(6) tag allows the fusion proteins purified in one step by immobilized metal (Ni(2+)) chelation affinity chromatography in the denatured or native state. As an example of the general utility of these expression vectors, the His(6)-fused catalytic subunits of mouse cAMP-dependent protein kinase were expressed with high activity by using these vectors and could be purified to homogeneity in one step.
Chen
Chang-Zheng
CZ
Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, China.
Huang
Hua
H
Gong
Jian
J
Xia
Qi-Chang
QC
Li
Bo-Liang
BL
Wang
Ying-Lai
YL