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

Pharmaceutics
Stefan VogtGordana Wozniak-Knopp

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 ...Continue Reading

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Methods Mentioned

BETA
transfection
Light Scattering
FCS
fluorescence-activated cell sorting
ELISA
Size Exclusion Chromatography

Software Mentioned

ASTRA
SWISS
MicroCal PEAQ - DSC Software
DSDBASE

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