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Pharmaceutics 2018, 10(3), 138; https://doi.org/10.3390/pharmaceutics10030138

Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting

1
acib GmbH (Austrian Centre of Industrial Biotechnology), Petersgasse 14, A-8010 Graz, Austria
2
Christian Doppler Laboratory for Innovative Immunotherapeutics, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, Austria
3
Christian Doppler Laboratory for Biotechnology of Skin Aging, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, Austria
4
Evercyte GmbH, Muthgasse 18, 1190 Wien, Austria
*
Author to whom correspondence should be addressed.
Received: 18 May 2018 / Revised: 21 August 2018 / Accepted: 22 August 2018 / Published: 27 August 2018
(This article belongs to the Special Issue Protein Therapeutics)
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Abstract

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. View Full-Text
Keywords: CD81 large extracellular loop; peptide grafting; antigen recognition unit; stability engineering CD81 large extracellular loop; peptide grafting; antigen recognition unit; stability engineering
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Vogt, S.; Stadlmayr, G.; Stadlbauer, K.; Sádio, F.; Andorfer, P.; Grillari, J.; Rüker, F.; Wozniak-Knopp, G. Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting. Pharmaceutics 2018, 10, 138.

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