Abstract
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. coli beta-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 Cerebratulus with 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 Hyp10 by 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 Pro10 may also contribute.
Highlights
Analysis of B-IV structureand function has identified Tyrg andTrp30as being essential for activity (7,s)
The complete covalent structures of two of the four Btoxins have been determined [3]. Both proteins are highly cationic and contain four disulfide bonds within their 55residue sequences. Despite this superficial similarity to other Na+-channel-selective toxinsderived from scorpion and anemone venoms, the Cerebratulus toxins display no amino acid sequence homology to any of these other polypeptides
A clone with the correct gene sequence, we decided to repair both mutations by oligonucleotide site-directed mutagenesisa.cid The mutated B-IV gene from clone112 was excised as an Aspartic Threonin e1 rrsiduc.s/rnol
Summary
Enzymes and Reagents-j3-Cyanoethylphosphoramidites were obtainedeither from PharmaciaLKB Biotechnology Inc. or ABN. M13 phage were passaged through E. coli dut- ung- strain BW313 [11]to prepare uracil-containing DNA templates for site-directed mutagenesis. E. coli strain BL21(DE3)isa X lysogen of BL21 wherein the prophage contains the RNA polymerase gene of T 7 bacteriophage under control of the hcUV5 promoter [12]. PSY1443 as an SphI/HindIII fragment and inserted into aderivative with rabbit antibody against B-IV This antibody, prepared from of plasmid pBR322 from which the SalI site hasbeen deleted to yield serum of rabbits injected with authentic B-IV and maintained on a2 the plasmid designated pSR9. Oligonucleotide-directed mutagenesis using uracil-containing single-stranded M13 templates andT4 DNA polymerase for second strand synthesis was performed as described by Kunkel et al [11].
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