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PEDS Advance Access originally published online on September 9, 2005
Protein Engineering Design and Selection 2005 18(11):515-526; doi:10.1093/protein/gzi057
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Kenji Fukuda1,2,3, Malene H. Jensen4, Richard Haser4, Nushin Aghajari4 and Birte Svensson1,2,5

1Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, 2Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads, Bldn. 224, DK-2800 Kgs. Lyngby, Denmark and 4Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086–CNRS/UCBL, IFR 128 ‘BioSciences Lyon-Gerland’, 7 Passage du Vercors, F-69367 Lyon cedex 07, France

5 To whom correspondence should be addressed, at the Technical University of Denmark. E-mail: bis{at}biocentrum.dtu.dk

Recombinant barley {alpha}-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure–function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14–Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a ß->{alpha} loop 2 that interacts with domain B (ß->{alpha} loop 3) protruding from the catalytic (ß/{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.

Keywords: codon usage/isozyme sequence-guided mutagenesis/stability/x-ray crystallography/yeast

Received April 27, 2005; revised August 5, 2005; accepted August 5, 2005.

Edited by Bauke Dijkstra


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[Abstract] [Full Text] [PDF]


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