| Abstrakt: |
A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli. Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E. coliβ-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient. The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture. The recombinant protein is identical with B-IV isolated from Cerebratuluswith respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10by Pro. Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity. The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10may also contribute. |
