Expressing and purifying membrane transport proteins in high yield.
Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.
Hale
Calvin C
CC
The Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA. chale@missouri.edu
Hill
Chananada K
CK
Price
Elmer M
EM
Bossuyt
Julie
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007814:Larva; D009036:Moths / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000302:isolation & purification*
D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1