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

Journal Article · · Protein Expression and Purification
 [1];  [2];  [3];  [4];  [5];  [6];  [7];  [8];  [7];  [9];  [10];  [10]
  1. Univ. of Copenhagen (Denmark); SLAC
  2. Univ. of Copenhagen (Denmark); Agilent Technologies, Glostrup (Denmark)
  3. Univ. of Copenhagen (Denmark); SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., CA (United States)
  4. Univ. of Copenhagen (Denmark); Univ. of Southern Denmark, Odense (Denmark)
  5. National Inst. for Biotechnology and Genetic Engineering (NIBGE), Faisalabad (Pakistan)
  6. Univ. of the Punjab, Lahore (Pakistan)
  7. Univ. of Copenhagen (Denmark). Novo Nordisk Foundation Center for Protein Research
  8. Nanotemper Technologies, Taastrup (Denmark); Symphogen A/S, Ballerup (Denmark)
  9. Aarhus Univ. (Denmark). DANDRITE, Nordic EMBL Partnership for Molecular Medicine; Aarhus Univ. (Denmark). Interdisciplinary Nanoscience Center (iNANO)
  10. Univ. of Copenhagen (Denmark)
+ Show Author Affiliations
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.
Research Organization:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Organization:
National Aeronautic and Space Administration (NASA); USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC02-76SF00515
OSTI ID:
1839400
Journal Information:
Protein Expression and Purification, Journal Name: Protein Expression and Purification Vol. 190; ISSN 1046-5928
Publisher:
ElsevierCopyright Statement
Country of Publication:
United States
Language:
English

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Rapid and Sensitive Quantification of Intracellular Glycyl-Sarcosine for Semi-High-Throughput Screening for Inhibitors of PEPT-1 journal July 2021

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Binding studies
Negative-stain electron microscopy
Peptide transporter
Protein expression
Protein purification
hPEPT1