Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1
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Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1. / Rafiq, Maria; Ernst, Heidi A.; Aduri, Nanda G.; Prabhala, Bala K.; Tufail, Soban; Rahman, Moazur; Bloch, Magnus Borup; Mirza, Nadia; Taylor, Nicholas M.; Boesen, Thomas; Gajhede, Michael; Mirza, Osman.
I: Protein Expression and Purification, Bind 190, 105990, 2022.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Expression, purification and characterization of human proton-coupled oligopeptide transporter 1 hPEPT1
AU - Rafiq, Maria
AU - Ernst, Heidi A.
AU - Aduri, Nanda G.
AU - Prabhala, Bala K.
AU - Tufail, Soban
AU - Rahman, Moazur
AU - Bloch, Magnus Borup
AU - Mirza, Nadia
AU - Taylor, Nicholas M.
AU - Boesen, Thomas
AU - Gajhede, Michael
AU - Mirza, Osman
PY - 2022
Y1 - 2022
N2 - 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 insolution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.
AB - 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 insolution oligomeric state of hPEPT1, negative stain electron microscopy was performed, demonstrating a predominantly monomeric state.
KW - Peptide transporter
KW - hPEPT1
KW - Protein expression
KW - Protein purification
KW - Binding studies
KW - Negative-stain electron microscopy
KW - BETA-LACTAM ANTIBIOTICS
KW - PEPTIDE TRANSPORTERS
KW - FAMILY SLC15
KW - DRUG
KW - RECOGNITION
KW - INHIBITORS
U2 - 10.1016/j.pep.2021.105990
DO - 10.1016/j.pep.2021.105990
M3 - Journal article
C2 - 34637915
VL - 190
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
M1 - 105990
ER -
ID: 283758042