A unique approach for high level expression and production of a recombinant cobra neurotoxin in Escherichia coli.
In this report, we describe a simple approach to produce a large quantity of a recombinant cobra neurotoxin containing four pairs of disulfide bonds. A cDNA encoding the toxin was fused, in frame, to the carboxyl termini of thioredoxin via a linker sequence encoding two amino acids, Asp and Pro. Due to the presence of thioredoxin, a soluble form of the fusion protein was expressed in a compartment, sensitive to osmotic pressure, in Escherichia coli. The fusion protein was released into the solution with low ionic strength under an osmotic shock treatment, and purified in a single step using an ion exchange chromatography column. The purified protein was treated in diluted hydrochloric acid to induce hydrolysis of the protein at the Asp-Pro linker site. Then, the recombinant neurotoxin was purified by gel filtration of the acid-treated sample. When the biological activity of the purified toxin was assayed, it was as potent as the natural toxin. Using this protocol, approximately 12 mg of pure recombinant neurotoxin can be produced from one liter of bacterial culture. More importantly, this protocol can be easily used for the production of the toxin at a larger scale with low cost. The approach outlined in this report will be suitable for the production of other recombinant proteins especially those of the 'three-finger' family.
Wang
Yongbao
Y
School of Life Science, University of Science and Technology of China, Hefei, Anhui province, People's Republic of China. ybwang@nicpbp.org.cn
Jing
Liu
L
Xu
Kangsen
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003039:Cobra Neurotoxin Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013879:Thioredoxins / Q000235:genetics
D003039:Cobra Neurotoxin Proteins; D004220:Disulfides; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins