Format

Send to

Choose Destination
Biochim Biophys Acta. 2000 Dec 29;1543(2):408-415.

The consensus concept for thermostability engineering of proteins.

Author information

1
F. Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals Division, Building 2411865, Basel, Switzerland.

Abstract

Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (T(m)) of 78.0 degrees C which is 15-22 degrees C higher than the T(m) values of all parent phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus phytases with T(m) values of up to 90.4 degrees C. These increases in T(m) are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37 degrees C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins.

PMID:
11150616
DOI:
10.1016/s0167-4838(00)00238-7
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center