Thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to S-citalopram

Jonathan A. Coleman, Evan M. Green, Eric Gouaux

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Original languageEnglish (US)
Article numbere54792
JournalJournal of Visualized Experiments
Volume2016
Issue number117
DOIs
StatePublished - Nov 27 2016

Fingerprint

Serotonin Plasma Membrane Transport Proteins
Citalopram
Crystallization
Antibodies
Purification
Sodium Chloride Symporters
Serotonin
Sf9 Cells
Immunoglobulin Fragments
Antibody Affinity
Baculoviridae
Sodium Chloride
Antidepressive Agents
Assays
Membrane Proteins
Anxiety
Sodium
X-Rays
Pumps
Depression

Keywords

  • Antibody
  • Antidepressant
  • Biochemistry
  • Crystallization
  • Immunization
  • Issue 117
  • Membrane protein
  • Neurotransmitter
  • Reconstitution
  • Selective serotonin reuptake inhibitor
  • Serotonin transporter
  • Structural biology
  • Thermostability

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Thermostabilization, expression, purification, and crystallization of the human serotonin transporter bound to S-citalopram. / Coleman, Jonathan A.; Green, Evan M.; Gouaux, Eric.

In: Journal of Visualized Experiments, Vol. 2016, No. 117, e54792, 27.11.2016.

Research output: Contribution to journalArticle

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