Change search
ReferencesLink to record
Permanent link

Direct link
Construction, expression and characterization of TEV protease mutants engineered for improved solubility
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0001-9504-4054
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.

Keyword [en]
proteases, flow cytometry, GFP, protein engineering, ssrA, ClpXP, tobacco etch virus, TEV, site-specific, activity, solubility, proteolysis
National Category
Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
URN: urn:nbn:se:kth:diva-33547OAI: oai:DiVA.org:kth-33547DiVA: diva2:416059
Funder
Swedish Research Council, 621-2004-4647
Note
QC 20110513Available from: 2011-05-13 Created: 2011-05-10 Last updated: 2011-05-16Bibliographically approved
In thesis
1. 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

Open Access in DiVA

fulltext(2286 kB)1120 downloads
File information
File name FULLTEXT01.pdfFile size 2286 kBChecksum SHA-512
82ffe1f4ee27ad6243521f86709bf234f7e1eb8da6435627b6e1df9a953f05018b032d501721789a1cc541cd81e6f62236b6812d3313ad24a869b822d28a95b1
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Kostallas, GeorgeSandersjöö, LisaAl-Askeri, MohammedSamuelson, Patrik
By organisation
Molecular Biotechnology
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1120 downloads
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

Total: 238 hits
ReferencesLink to record
Permanent link

Direct link