Expression of neurotransmitter transporters for structural and biochemical studies.
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.
Elbaz
Yael
Y
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem, Israel.
Danieli
Tsafi
T
Kanner
Baruch I
BI
Schuldiner
Shimon
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001692:Biological Transport; D002474:Cell-Free System; D002478:Cells, Cultured; D019085:Fluorescent Antibody Technique, Direct; D005822:Genetic Vectors; D007150:Immunohistochemistry; D026901:Membrane Transport Proteins / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D050479:Neurotransmitter Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology; D050493:Vesicular Monoamine Transport Proteins / Q000302:isolation & purification / Q000378:metabolism
D026901:Membrane Transport Proteins; D018377:Neurotransmitter Agents; D050479:Neurotransmitter Transport Proteins; D050493:Vesicular Monoamine Transport Proteins