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PEDS Advance Access published online on December 1, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh091
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Received August 30, 2004
Revised November 16, 2004
Accepted November 19, 2004

Article

Construction of stabilized proteins by combinatorial consensus mutagenesis

N. Amin 1, A. D. Liu 1, S. Ramer 1, W. Aehle 2, D. Meijer 2, M. Metin 2, S. Wong 1, P. Gualfetti 1, and V. Schellenberger 1*

1 Genencor International, 925 Page Mill Road, Palo Alto, CA 94304
2 Genencor International b.v. Leiden, The Netherlands

* To whom correspondence should be addressed.
V. Schellenberger, E-mail: vschellenberger{at}genencor.com


  Abstract

We constructed stabilized variants of {beta}-lactamase (BLA) from E. cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E. cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and 8 mutations that destabilized the protein. A second generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees higher than the starting molecule and contained 8 consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.

Keywords: thermostability; proteolysis; lactamase; consensus mutation; combinatorial mutagenesis.
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