Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting
Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal...
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MDPI AG
2018-08-01
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| Series: | Pharmaceutics |
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| Online Access: | http://www.mdpi.com/1999-4923/10/3/138 |
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| author | Stefan Vogt Gerhard Stadlmayr Katharina Stadlbauer Flávio Sádio Peter Andorfer Johannes Grillari Florian Rüker Gordana Wozniak-Knopp |
| author_facet | Stefan Vogt Gerhard Stadlmayr Katharina Stadlbauer Flávio Sádio Peter Andorfer Johannes Grillari Florian Rüker Gordana Wozniak-Knopp |
| author_sort | Stefan Vogt |
| collection | DOAJ |
| description | Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold. |
| first_indexed | 2024-04-11T21:41:12Z |
| format | Article |
| id | doaj.art-87f2b8d2374e4b10bd2d37e083e8ef0f |
| institution | Directory Open Access Journal |
| issn | 1999-4923 |
| language | English |
| last_indexed | 2024-04-11T21:41:12Z |
| publishDate | 2018-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pharmaceutics |
| spelling | doaj.art-87f2b8d2374e4b10bd2d37e083e8ef0f2022-12-22T04:01:35ZengMDPI AGPharmaceutics1999-49232018-08-0110313810.3390/pharmaceutics10030138pharmaceutics10030138Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide GraftingStefan Vogt0Gerhard Stadlmayr1Katharina Stadlbauer2Flávio Sádio3Peter Andorfer4Johannes Grillari5Florian Rüker6Gordana Wozniak-Knopp7acib GmbH (Austrian Centre of Industrial Biotechnology), Petersgasse 14, A-8010 Graz, AustriaChristian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, AustriaChristian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, AustriaChristian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, AustriaChristian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, AustriaChristian Doppler Laboratory for Biotechnology of Skin Aging, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, Austriaacib GmbH (Austrian Centre of Industrial Biotechnology), Petersgasse 14, A-8010 Graz, Austriaacib GmbH (Austrian Centre of Industrial Biotechnology), Petersgasse 14, A-8010 Graz, AustriaTetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.http://www.mdpi.com/1999-4923/10/3/138CD81 large extracellular looppeptide graftingantigen recognition unitstability engineering |
| spellingShingle | Stefan Vogt Gerhard Stadlmayr Katharina Stadlbauer Flávio Sádio Peter Andorfer Johannes Grillari Florian Rüker Gordana Wozniak-Knopp Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting Pharmaceutics CD81 large extracellular loop peptide grafting antigen recognition unit stability engineering |
| title | Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting |
| title_full | Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting |
| title_fullStr | Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting |
| title_full_unstemmed | Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting |
| title_short | Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting |
| title_sort | stabilization of the cd81 large extracellular loop with de novo disulfide bonds improves its amenability for peptide grafting |
| topic | CD81 large extracellular loop peptide grafting antigen recognition unit stability engineering |
| url | http://www.mdpi.com/1999-4923/10/3/138 |
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