uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
An improved dual-tube megaprimer approach for multi-site saturation mutagenesis
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
2013 (English)In: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 29, no 4, 667-672 p.Article in journal (Refereed) Published
Abstract [en]

Saturation mutagenesis is a powerful tool in protein engineering. Even though QuikChange site-directed mutagenesis method is dominantly used in laboratories, it could not be successfully applied to the generation of a focused mutant library of human glutathione transferase A2-2. In the present study, we further developed an improved versatile dual-tube approach of randomizing difficult-to-amplify targets, exhibiting significant improvement towards equal distribution of nucleotides at randomized sites compared to other published methods.

Place, publisher, year, edition, pages
2013. Vol. 29, no 4, 667-672 p.
National Category
Natural Sciences
URN: urn:nbn:se:uu:diva-158396DOI: 10.1007/s11274-012-1222-zISI: 000316291000010OAI: oai:DiVA.org:uu-158396DiVA: diva2:439216
Available from: 2011-09-06 Created: 2011-09-06 Last updated: 2013-04-22Bibliographically approved
In thesis
1. Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
Open this publication in new window or tab >>Directed Evolution of Glutathione Transferases with Altered Substrate Selectivity Profiles: A Laboratory Evolution Study Shedding Light on the Multidimensional Nature of Epistasis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Directed evolution is generally regarded as a useful approach in protein engineering. By subjecting members of a mutant library to the power of Darwinian evolution, desired protein properties are obtained. Numerous reports have appeared in the literature showing the success of tailoring proteins for various applications by this method. Is it a one-way track that protein practitioners can only learn from nature to enable more efficient protein engineering?

A structure-and-mechanism-based approach, supplemented with the use of reduced amino acid alphabets, was proposed as a general means for semi-rational enzyme engineering. Using human GST A2-2*E, the most active human enzyme in the bioactivation of azathioprine, as a parental enzyme to test this approach, a L107G/L108D/F222H triple-point mutant of GST A2-2*E (thereafter designated as GDH) was discovered with 70-fold increased activity, approaching the upper limit of specific activity of the GST scaffold. The approach was further experimentally verified to be more successful than intuitively choosing active-site residues in proximity to the bound substrate for the improvement of enzyme performance.

By constructing all intermediates along all putative mutational paths leading from GST A2-2*E to mutant GDH and assaying them with nine alternative substrates, the fitness landscapes were found to be “rugged” in differential fashions in substrate-activity space. The multidimensional fitness landscapes stemming from functional promiscuity can lead to alternative outcomes with enzymes optimized for other features than the selectable markers that were relevant at the origin of the evolutionary process. The results in this thesis suggest that in this manner an evolutionary response to changing environmental conditions can readily be mounted.

In summary, the thesis demonstrates the attractive features of the structure-and-mechanism-based semi-rational directed evolution approach for optimizing enzyme performance. Moreover, the results gained from the studies show that laboratory evolution may refine our understanding of evolutionary process in nature.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 47 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 850
glutathione transferase, azathioprine, directed evolution, semi-rational design, catalytic mechanism, saturation mutagenesis, reduced amino acid alphabets, molecular docking, protein evolution, multivariate data analysis, epistasis, fitness landscape, evolutionary trajectories
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:uu:diva-158400 (URN)978-91-554-8147-6 (ISBN)
Public defence
2011-10-21, C2:301, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2011-09-30 Created: 2011-09-06 Last updated: 2011-11-03Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Zhang, WeiMannervik, Bengt
By organisation
In the same journal
World Journal of Microbiology & Biotechnology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 435 hits
ReferencesLink to record
Permanent link

Direct link