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 Scopus citations

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

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

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