Expression and purification of human TRPV1 in baculovirus-infected insect cells for structural studies.
TRPV1 is a ligand-gated cation channel that is involved in acute thermal nociception and neurogenic inflammation. By using the GP67 signal peptide, high levels of full-length human TRPV1 was expressed in High Five insect cells using the baculovirus expression system. The functional activity of the expressed TRPV1 was confirmed by whole-cell ligand-gated ion flux recordings in the presence of capsaicin and low pH and via specific ligand binding to the isolated cellular membranes. Efficient solubilization and purification protocols have resulted in milligram amounts of detergent-solubilized channel at 80-90% purity after Ni2+ IMAC chromatography and size exclusion chromatography. Western blot analysis of amino and carboxyl terminal domains and MS of tryptic digestions of purified protein confirmed the presence of the full-length human TRPV1. Specific ligand binding experiments confirmed the protein integrity of the purified human TRPV1.
Korepanova
Alla
A
Department of Structural Biology, R46Y, Abbott Laboratories, Bldg. AP10-LL8, 100 Abbott Park Rd., Abbott Park, IL 60064-6098, USA. alla.korepanova@abbott.com
Pereda-Lopez
Ana
A
Solomon
Larry R
LR
Walter
Karl A
KA
Lake
Marc R
MR
Bianchi
Bruce R
BR
McDonald
Heath A
HA
Neelands
Torben R
TR
Shen
Jianwei
J
Matayoshi
Edmund D
ED
Moreland
Robert B
RB
Chiu
Mark L
ML
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D015870:Gene Expression; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D018411:Spodoptera; D050916:TRPV Cation Channels / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human