PMID- 20399272
OWN - NLM
STAT- MEDLINE
DCOM- 20101207
LR  - 20211020
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Print)
IS  - 1046-5928 (Linking)
VI  - 74
IP  - 1
DP  - 2010 Nov
TI  - Functional expression, purification and high sequence coverage mass spectrometric 
      characterization of human excitatory amino acid transporter EAAT2.
PG  - 49-59
LID - 10.1016/j.pep.2010.04.006 [doi]
AB  - The glial excitatory amino acid transporter 2 (EAAT2) mediates a majority of 
      glutamate re-uptake in human CNS and, consequently, is associated with a variety 
      of signaling and pathological processes. While our understanding of the function, 
      mechanism and structure of this integral membrane protein is increasing, little 
      if any mass spectrometric (MS) data is available for any of the EAATs 
      specifically, and for only a few mammalian plasma membrane transporters in 
      general. A protocol to express and purify functional EAAT2 in sufficient 
      quantities to carry out MS-based peptide mapping as needed to study 
      ligand-transporter interactions is described. A 6xHIS epitope was incorporated 
      into the N-terminus of human EAAT2. The recombinant protein was expressed in high 
      levels in mammalian HEK 293T cells, where it exhibited the pharmacological 
      properties of the native transporter. EAAT2 was purified from isolated cell 
      membranes in a single step using nickel affinity chromatography. In-gel and 
      in-solution trypsin digestions were conducted on the isolated protein and then 
      analyzed by MALDI-TOF and LC-MS/MS mass spectrometry. Overall, 89% sequence 
      coverage of the protein was achieved with these methods. In particular, an 88 
      amino acid tryptic peptide covering the presumed substrate binding domains HP1, 
      TMD7, HP2, and TMD8 domains of EAAT2 was also identified after N-deglycosylation. 
      Beyond the specific applicability to EAAT2, this study provides an efficient, 
      simple and scalable approach to express, purify, digest and characterize integral 
      membrane transporter proteins by mass spectrometry.
CI  - Copyright 2010. Published by Elsevier Inc.
FAU - Ye, Ran
AU  - Ye R
AD  - Center for Structural and Functional Neuroscience, Department of Chemistry and 
      Biochemistry, The University of Montana, Missoula, MT 59812, USA.
FAU - Rhoderick, Joseph F
AU  - Rhoderick JF
FAU - Thompson, Charles M
AU  - Thompson CM
FAU - Bridges, Richard J
AU  - Bridges RJ
LA  - eng
GR  - P20 RR015583/RR/NCRR NIH HHS/United States
GR  - R01 NS030570/NS/NINDS NIH HHS/United States
GR  - P20 RR15583/RR/NCRR NIH HHS/United States
GR  - NS30570/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20100422
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Excitatory Amino Acid Transporter 2)
RN  - EC 3.4.21.4 (Trypsin)
SB  - IM
MH  - Amino Acid Sequence
MH  - Cell Line
MH  - Cell Membrane/metabolism
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Excitatory Amino Acid Transporter 2/*chemistry/*genetics/isolation & 
      purification/metabolism
MH  - *Gene Expression
MH  - Humans
MH  - Molecular Sequence Data
MH  - Solubility
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH  - Trypsin/metabolism
PMC - PMC4356123
MID - NIHMS666441
EDAT- 2010/04/20 06:00
MHDA- 2010/12/14 06:00
PMCR- 2015/03/11
CRDT- 2010/04/20 06:00
PHST- 2010/03/05 00:00 [received]
PHST- 2010/04/08 00:00 [revised]
PHST- 2010/04/09 00:00 [accepted]
PHST- 2010/04/20 06:00 [entrez]
PHST- 2010/04/20 06:00 [pubmed]
PHST- 2010/12/14 06:00 [medline]
PHST- 2015/03/11 00:00 [pmc-release]
AID - S1046-5928(10)00110-5 [pii]
AID - 10.1016/j.pep.2010.04.006 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2010 Nov;74(1):49-59. doi: 10.1016/j.pep.2010.04.006. Epub 
      2010 Apr 22.