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Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
2011 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 1, e16136- p.Article in journal (Refereed) Published
Abstract [en]

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P19, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.

Place, publisher, year, edition, pages
2011. Vol. 6, no 1, e16136- p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-31599DOI: 10.1371/journal.pone.0016136ISI: 000286519500041Scopus ID: 2-s2.0-79251539925OAI: oai:DiVA.org:kth-31599DiVA: diva2:406325
Funder
Swedish Research Council, 621-2004-4647
Note
QC 20110325Available from: 2011-03-25 Created: 2011-03-21 Last updated: 2011-05-16Bibliographically 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009: 12
Keyword
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
Identifiers
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)
Opponent
Supervisors
Note
QC 20100729Available from: 2009-09-08 Created: 2009-09-02 Last updated: 2011-12-08Bibliographically approved
2. Intracellular systems for characterization and engineering of proteases and their substrates
Open this publication in new window or tab >>Intracellular systems for characterization and engineering of proteases and their substrates
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention.

With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media.

By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III).

We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. x, 52 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:11
Keyword
proteases, flow cytometry, GFP, CAT, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, high-throughput, selection, proteolysis, libraries
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-33549 (URN)978-91-7415-992-9 (ISBN)
Public defence
2011-05-31, Lecture hall FD5 (Svedbergsalen) AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 621-2004-4647
Note
QC 20110516Available from: 2011-05-16 Created: 2011-05-10 Last updated: 2011-05-16Bibliographically approved

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