Expression of a Synthetic Mussel Adhesive Protein in Escherichia Coli
Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli. The bioadhesive precursor (BP) with a MW of 25,000 was expressed from one 600-bp gene comprised of a 30-bp unit repeat that accounts for E. coli codon bias. In strains employing T7 RNA polymerase for induction, BP was produced at levels approaching 60% of total cell protein. BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein as shown by amino acid composition and N-terminal sequencing to give an authentic consensus precursor protein. Although the repetitious gene containing 30-bp repeat units appeared stable in T7-based host/vector systems, it was less stable in a λPL promoter-based host/vector system. Codon diversification was examined as a potential method to alleviate the problems by constructing a repetitious gene comprised of 120-bp repeats. This longer repeat unit failed to confer additional stability upon the repetitious gene.
KeywordsRepeat Unit Cetyl Trimethyl Ammonium Bromide Mytilus Edulis Gene Cassette Total Cell Protein
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