期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 117, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/54792
关键词
Biochemistry; Issue 117; structural biology; crystallization; membrane protein; antibody; immunization; reconstitution; serotonin transporter; neurotransmitter; antidepressant; selective serotonin reuptake inhibitor; thermostability
资金
- Banting postdoctoral fellowship
- Canadian Institutes of Health Research
- National Science Foundation Graduate Research Fellowship
- NIH [5R37MH070039]
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 angstrom resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.
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