Abstract
Neurotransmitter transporters play essential roles in the process of neurotransmission. Vesicular neurotransmitter transporters mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H+ for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter. Our current understanding of the mechanism of these transporters is based on functional and biochemical characterization but is lacking high-resolution structural information. Very few structures of membrane transport systems from mammalian origin have been solved to atomic resolution, mainly because of the difficulty in obtaining large amounts of purified protein. Development of high yield heterologous expression systems suitable for mammalian neurotransmitter transporters is essential to enable the production of purified protein for structural studies. Such a system makes possible also the production of mutants that can be used in biochemical and biophysical studies. We describe here a screen for the expression of the vesicular monoamine transporter 2 (VMAT2) in cell-free and baculovirus expression systems and discuss the expression of VMAT2 in other systems as well (bacterial, yeast and mammalian cell lines). After screening and optimization, we achieved high yield (2-2.5 mg/l) expression of functional VMAT2 in insect cells. The system was also used for the expression of three additional plasma membrane neurotransmitter transporters. All were functional and expressed to high levels. Our results demonstrate the advantages of the baculovirus expression system for the expression of mammalian neurotransmitter transporters in a functional state.
| Original language | English |
|---|---|
| Pages (from-to) | 152-160 |
| Number of pages | 9 |
| Journal | Protein Expression and Purification |
| Volume | 73 |
| Issue number | 2 |
| DOIs | |
| State | Published - Oct 2010 |
Bibliographical note
Funding Information:We wish to thank Etay Benovich for assistance and guidance in the very early stages of work with the baculovirus expression system, Hila Frenkel for excellent technical support regarding cell-free expression systems, Dr. Mario Lebendiker from the protein purification facility for his excellent guidance and advice in the purification of VMAT2, Shmulik Ittah and Dr. Naomi Melamed-Book for assistance in preparing and analyzing samples using confocal microscopy, Sonia Steiner-Mordoch for assistance in screening VMAT2 expression in bacterial strains and Annie Bendahan for sub-cloning GAT1, EAAC1 and GLT1 into pVL1393 and for the reconstitution of these proteins. Y.E. is supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities. This work was supported by Grants from the Israel Science Foundation (BIK), the European Union Consortium European Drug Initiative on Channels and Transporters (BIK) and the National Institute of Health (NS16708 to SS) and by The Center for Innovation in Membrane Protein Production (P50 GM73210). SS is Mathilda Marks-Kennedy Professor of Biochemistry at the Hebrew University of Jerusalem.
Keywords
- Baculovirus
- Cell-free expression
- Insect cells
- Membrane proteins
- VMAT
