Motif-driven protein binder design towards transferrin receptor helical domain
© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies..
Human transferrin receptor 1 (TfR) is necessary for the delivery of the iron carrier protein transferrin into cells and can be utilized for targeted delivery across cellular membranes. Binding of transferrin to the receptor is regulated by hereditary hemochromatosis protein (HFE), an iron regulatory protein that partly shares a binding site with transferrin on TfR. Here, we derived essential binding interactions from HFE and computationally grafted these into a library of small protein scaffolds. One of the designed proteins, TB08, was further optimized computationally and experimentally to identify variants with improved binding to TfR. The optimized variant, TB08 S3.1, expressed well in the E. coli expression system and had an affinity to TfR in the low micromolar range, Kd ≈ 1 μm, as determined by surface plasmon resonance. A binding competition assay with transferrin further confirmed the interaction of the evolved variant to TfR at the shared binding surface. Additionally, the GFP-tagged evolved variant of TB08 demonstrated cellular internalization as determined by fluorescent and confocal microscopy in HeLa cells. The designed protein is small, allows for robust cargo tagging, and interacts specifically with TfR, thus making it a valuable tool for the characterization of TfR-mediated cellular transport mechanisms and for the assessment of engineering strategies for cargo delivery across cell membranes.
Medienart: |
E-Artikel |
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Erscheinungsjahr: |
2022 |
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Erschienen: |
2022 |
Enthalten in: |
Zur Gesamtaufnahme - volume:289 |
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Enthalten in: |
The FEBS journal - 289(2022), 10 vom: 20. Mai, Seite 2935-2947 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Sjöström, Dick J [VerfasserIn] |
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Links: |
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Themen: |
Journal Article |
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Anmerkungen: |
Date Completed 19.05.2022 Date Revised 15.06.2022 published: Print-Electronic Citation Status MEDLINE |
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doi: |
10.1111/febs.16311 |
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funding: |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
NLM333994868 |
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520 | |a © 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. | ||
520 | |a Human transferrin receptor 1 (TfR) is necessary for the delivery of the iron carrier protein transferrin into cells and can be utilized for targeted delivery across cellular membranes. Binding of transferrin to the receptor is regulated by hereditary hemochromatosis protein (HFE), an iron regulatory protein that partly shares a binding site with transferrin on TfR. Here, we derived essential binding interactions from HFE and computationally grafted these into a library of small protein scaffolds. One of the designed proteins, TB08, was further optimized computationally and experimentally to identify variants with improved binding to TfR. The optimized variant, TB08 S3.1, expressed well in the E. coli expression system and had an affinity to TfR in the low micromolar range, Kd ≈ 1 μm, as determined by surface plasmon resonance. A binding competition assay with transferrin further confirmed the interaction of the evolved variant to TfR at the shared binding surface. Additionally, the GFP-tagged evolved variant of TB08 demonstrated cellular internalization as determined by fluorescent and confocal microscopy in HeLa cells. The designed protein is small, allows for robust cargo tagging, and interacts specifically with TfR, thus making it a valuable tool for the characterization of TfR-mediated cellular transport mechanisms and for the assessment of engineering strategies for cargo delivery across cell membranes | ||
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650 | 4 | |a protein design | |
650 | 4 | |a transferrin receptor | |
650 | 4 | |a yeast surface display | |
650 | 7 | |a Membrane Proteins |2 NLM | |
650 | 7 | |a Receptors, Transferrin |2 NLM | |
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700 | 1 | |a Mohlin, Camilla |e verfasserin |4 aut | |
700 | 1 | |a Ambrosetti, Elena |e verfasserin |4 aut | |
700 | 1 | |a Garforth, Scott J |e verfasserin |4 aut | |
700 | 1 | |a Teixeira, Ana I |e verfasserin |4 aut | |
700 | 1 | |a Bjelic, Sinisa |e verfasserin |4 aut | |
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