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

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.

[1]  F. Arnold.  Combinatorial and computational challenges for biocatalyst design , 2001, Nature.

[2]  B. Dahiyat,et al.  In silico design for protein stabilization. , 1999, Current opinion in biotechnology.

[3]  A. Fersht,et al.  Design of highly stable functional GroEL minichaperones , 1999, Protein science : a publication of the Protein Society.

[4]  Stephen L. Mayo,et al.  Design, structure and stability of a hyperthermophilic protein variant , 1998, Nature Structural Biology.

[5]  F. Arnold,et al.  Directed evolution converts subtilisin E into a functional equivalent of thermitase. , 1999, Protein engineering.

[6]  Jon E. Ness,et al.  DNA shuffling of subgenomic sequences of subtilisin , 1999, Nature Biotechnology.

[7]  F. Arnold,et al.  Designed evolution of enzymatic properties. , 2000, Current opinion in biotechnology.

[8]  J. Kelly,et al.  Increasing protein stability using a rational approach combining sequence homology and structural alignment: Stabilizing the WW domain , 2001, Protein science : a publication of the Protein Society.

[9]  F. Arnold,et al.  Directed evolution study of temperature adaptation in a psychrophilic enzyme. , 2000, Journal of molecular biology.

[10]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[11]  G. Fink,et al.  Laboratory course manual for methods in yeast genetics , 1986 .

[12]  G. Fink,et al.  Transformation of yeast. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Davidson,et al.  Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects. , 1998, Biochemistry.

[14]  G. Vriend,et al.  Evolution reversed: engineering and enzyme to resist boiling , 1998 .

[15]  B Steipe,et al.  Evolutionary approaches to protein engineering. , 1999, Current topics in microbiology and immunology.

[16]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[17]  M. Lehmann,et al.  From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. , 2000, Protein engineering.

[18]  A. Plückthun,et al.  Beyond binding: using phage display to select for structure, folding and enzymatic activity in proteins. , 1999, Current opinion in structural biology.

[19]  G Vriend,et al.  Protein stabilization by hydrophobic interactions at the surface. , 1994, European journal of biochemistry.

[20]  F. Arnold,et al.  Directed evolution of a thermostable esterase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  S. Steinbacher,et al.  Sequence statistics reliably predict stabilizing mutations in a protein domain. , 1994, Journal of molecular biology.

[22]  L Pasamontes,et al.  Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three‐dimensional structure , 2000, Protein science : a publication of the Protein Society.

[23]  J. Barnikow,et al.  Intrabody construction and expression. II. A synthetic catalytic Fv fragment. , 1999, Journal of Molecular Biology.

[24]  E Ohage,et al.  Intrabody construction and expression. I. The critical role of VL domain stability. , 1999, Journal of molecular biology.

[25]  G. Vriend,et al.  Engineering an enzyme to resist boiling. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Kurt Vogel,et al.  Engineering of Phytase for Improved Activity at Low pH , 2002, Applied and Environmental Microbiology.

[27]  A. Fersht,et al.  Stabilization of GroEL minichaperones by core and surface mutations. , 2000, Journal of molecular biology.

[28]  S. Steinbacher,et al.  β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability , 1997, Protein science : a publication of the Protein Society.

[29]  M. Wyss,et al.  Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens , 2001, Applied and Environmental Microbiology.

[30]  W. Zimmermann,et al.  The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae , 1990, Current Genetics.

[31]  Gerd Folkers,et al.  Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling , 1999, Nature Biotechnology.

[32]  F. Arnold,et al.  Temperature adaptation of enzymes: lessons from laboratory evolution. , 2000, Advances in protein chemistry.

[33]  A. Fersht,et al.  Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Hoseki,et al.  Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus. , 1999, Journal of biochemistry.

[35]  F. Arnold,et al.  A structural view of evolutionary divergence. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[36]  P. Wirtz,et al.  Intrabody construction and expression III: Engineering hyperstable VH domains , 2008, Protein science : a publication of the Protein Society.