Expression of Kappa Bungarotoxin in Escherichia Coli: Mutagenesis of a Fusion Protein Gene
Harmsel, Alyssa Ten
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A gene coding for a kappa bungarotoxin (K-Bgt) and rat intestinal fatty acid binding fusion protein was previously constructed in an Escherichia coli plasmid in order to allow synthesis of K-Bgt in vitro. This gene was site-specifically modified through the technique of cassette mutagenesis to remove the DNA sequence coding for a Factor Xa protease cleavage site. A synthetic sequence of double-stranded DNA coding for a methionine residue was inserted in its place. Thus, the resulting protein no longer contains the Factor Xa cleavage site, but instead contains methionine, which is subject to cyanogen bromide cleavage. A methionine residue between the two halves of the fusion protein will allow separation of intact K-Bgt from the fusion protein, since methionine is not present in the primary amino acid sequence of K-Bgt. Sequencing of the plasmid DNA after cassette mutagenesis had been performed confirmed that the insert containing the methionine-coding DNA was present. After induction and isolation of the fusion protein, cyanogen bromide cleavage was performed. Amino acid sequence analysis and immunoblotting assays showed that the correct fusion protein was obtained. Amino acid sequencing also showed that cyanogen bromide sequencing was successful, resulting in the intact kappa bungarotoxin and fragments of the rat intestinal fatty acid binding protein.