Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1

Maria Rafiq, Heidi A. Ernst, Nanda G. Aduri, Bala K. Prabhala, Soban Tufail, Moazur Rahman, Magnus Borup Bloch, Nadia Mirza, Nicholas M.I. Taylor, Thomas Boesen, Michael Gajhede, Osman Mirza*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

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Abstract

The human peptide transporter hPEPT1 (SLC15A1) is responsible for uptake of dietary di- and tripeptides and a number of drugs from the small intestine by utilizing the proton electrochemical gradient, and hence an important target for peptide-like drug design and drug delivery. hPEPT1 belongs to the ubiquitous major facilitator superfamily that all contain a 12TM core structure, with global conformational changes occurring during the transport cycle. Several bacterial homologues of these transporters have been characterized, providing valuable insight into the transport mechanism of this family. Here we report the overexpression and purification of recombinant hPEPT1 in a detergent-solubilized state. Thermostability profiling of hPEPT1 at different pH values revealed that hPEPT1 is more stable at pH 6 as compared to pH 7 and 8. Micro-scale thermophoresis (MST) confirmed that the purified hPEPT1 was able to bind di- and tripeptides respectively. To assess the in-solution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.

Original languageEnglish
Article number105990
JournalProtein Expression and Purification
Volume190
ISSN1046-5928
DOIs
Publication statusPublished - Feb 2022

Keywords

  • Binding studies
  • hPEPT1
  • Negative-stain electron microscopy
  • Peptide transporter
  • Protein expression
  • Protein purification

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