The consensus concept for thermostability engineering of proteins: further proof of concept

Abstract

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.

Designed to be stable: Crystal structure of a consensus ankyrin repeat protein

“…The canonical sequence approximation has previously been used to create molecules with improved stabilities (9,(11)(12)(13)(14). We have successfully applied this consensus design to ARs.…”

“…The collection of nearly 8,000 AR sequences in the SMART database (10) facilitates such an approach. This consensus design concept has been used to generate enzymes with improved thermostability (11,12) and to improve antibody stability (9,13,14). We used this concept to design a consensus AR consisting of fixed framework residues that are responsible for repeat structure maintenance (fold conservation) and of randomized interacting residues, i.e., residues, which, in a repeat domain, create a randomized surface for interaction with target proteins.…”

ProteinParser—A community based tool for the generation of a detailed protein consensus and FASTA output

“…By applying a consensus-based approach, substitute amino acids can be selected simply and rapidly, at low cost and often resulting in the generation of a stabilised protein ( [8], and references within). Quite simply, the "in silico" consensus protein dictates the replacement residues based on direct comparison between amino acids that deviate from the consensus protein.…”

“…This semi-rational protein design methodology predicts potential stabilising mutations in silico, which can be validated experimentally by site directed mutagenesis. Several reports of successful protein stabilisation employing this approach are noted in the literature [7][8][9]. The consensus approach is based on the assumption that conserved residues, as detailed from sequence alignments of related proteins, contribute more to the stabilisation of the protein than nonconserved residues [10].…”

Heterotachy and Functional Shift in Protein Evolution

“…MSA have efficiently been used to predict secondary structure of RNAs and proteins (32,33). The consensus sequence of fungal phytase inferred from MSA showed an increased thermostability (34). However, these methods do not make use of evolutionary information, i.e.…”