Construction of stabilized proteins by combinatorial consensus mutagenesis

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.

Selection and optimization of proteolytically stable llama single-domain antibody fragments for oral immunotherapy

“…Possibly, the higher proteolytic stability of the newly selected VHHs is similarly caused by reduced flexibility of CDR loops. Such a suggestion is in accordance with earlier observations that proteolytic and thermal stability of proteins generally are correlated because unfolded proteins expose flexible regions that are more sensitive to proteolysis (Arnold and Ulbrich-Hofmann 1997;Amin et al 2004). Furthermore, the proteolytic stabilization of certain enzymes by protein engineering was due to reduced flexibility of the protein region that was prone to proteolysis (Frenken et al 1993;Markert et al 2001).…”

Consensus mutagenesis reveals that non-helical regions influence thermal stability of horseradish peroxidase

“…Only two mutants registered marginal stability gains over wild type recombinant HRP, in sharp contrast to previous consensus studies with other proteins [19][20][21][22][24][25][26].…”

“…Unlike previous successful consensus studies with other proteins [19][20][21][22][24][25][26], none of the five substitutions yielded a significant gain in HRP thermal stability. Indeed, our consensus mutations had a greater influence on K' m for ABTS than on thermal stability (Table 1) In previous studies, six out of ten [19] and six out of twelve [46] consensus mutations were thermostabilizing; it has also been noted that thermostabilization occurs in about 33% of the total consensus mutants generated [22].…”

New insight into long‐range nonadditivity within protein double‐mutant cycles

“…Understanding the origins of nonadditivity is crucial for elucidating fundamental mechanisms of protein function such as cooperativity and allostery (6,7). The ability to predict the occurrence of nonadditivity is important for biotechnology applications such as protein stabilization (8,9) and protein design (10,11). It is well known that changes in functional properties within double mutant cycles are usually nonadditive when the mutated residues are in direct contact with each other [for review, see (12)].…”