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Improved solubility of TEV protease by directed evolution
KTH, School of Engineering Sciences (SCI), Physics. (Structural Biochemistry)
2006 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 121, no 3, 291-298 p.Article in journal (Refereed) Published
Abstract [en]

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV–GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEVSH, in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Place, publisher, year, edition, pages
Amsterdam: Elsevier , 2006. Vol. 121, no 3, 291-298 p.
Keyword [en]
TEV protease; Directed evolution; GFP; Solubility; FACS
National Category
Biocatalysis and Enzyme Technology Other Industrial Biotechnology
URN: urn:nbn:se:kth:diva-10962DOI: 10.1016/j.jbiotec.2005.08.006ISI: 000235504800001ScopusID: 2-s2.0-30944459888OAI: diva2:233309
QC 20100729Available from: 2009-09-02 Created: 2009-08-31 Last updated: 2010-12-06Bibliographically approved
In thesis
1. On bacterial formats in protein library technology
Open this publication in new window or tab >>On bacterial formats in protein library technology
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii,102 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2009: 12
Affibody molecule, β-lactamase, combinatorial library, green fluorescent protein (GFP), protein engineering, protein fragment complementation assay (PCA), selection, TEVprotease, TNF-α
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology
urn:nbn:se:kth:diva-10993 (URN)978-91-7415-400-9 (ISBN)
Public defence
2009-09-25, AlbaNova Room FA 32, Roslagstullsbacken 21, Stockholm, 10:00 (Swedish)
QC 20100729Available from: 2009-09-08 Created: 2009-09-02 Last updated: 2011-12-08Bibliographically approved

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