Construction of stabilized proteins by combinatorial consensus mutagenesis.
Protein Eng Des Sel
; 17(11): 787-93, 2004 Nov.
Article
em En
| MEDLINE
| ID: mdl-15574484
ABSTRACT
We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter 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 eight 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 C higher than the starting molecule and contained eight 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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Engenharia de Proteínas
/
Proteínas
/
Mutagênese
/
Técnicas de Química Combinatória
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article