Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast

Kenji Fukuda, Malene Hillerup Jensen, Nushin Aghajari, Richard Haser, Birte Svensson

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    Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation.
    Original languageEnglish
    JournalProtein Engineering Design and Selection (Print)
    Issue number11
    Pages (from-to)515-526
    Publication statusPublished - Nov 2005


    • codon usage
    • isozyme sequence-guided mutagenesis
    • stability
    • x-ray crystallography
    • yeast

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