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In-vitro selection of highly stabilized protein variants with optimized surface

Title data

Martin, Andreas ; Sieber, Volker ; Schmid, Franz X.:
In-vitro selection of highly stabilized protein variants with optimized surface.
In: Journal of Molecular Biology. Vol. 309 (8 June 2001) Issue 3 . - pp. 717-726.
ISSN 0022-2836
DOI: https://doi.org/10.1006/jmbi.2001.4698

Abstract in another language

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 11397091
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Professorship Biochemistry - Univ.-Prof. Dr. Franz Xaver Schmid
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Professorship Biochemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 28 May 2015 07:03
Last Modified: 28 May 2015 07:03
URI: https://eref.uni-bayreuth.de/id/eprint/14370