Expression of Kappa Bungarotoxin in Escherichia Coli: Mutagenesis of a Fusion Protein Gene
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Authors
Harmsel, Alyssa Ten
Issue Date
1990
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
vi, 36 p.
Citation
Publisher
Kalamazoo College
License
U.S. copyright laws protect this material. Commercial use or distribution of this material is not permitted without prior written permission of the copyright holder.