Figure 2.

Purification of NgBR–hCIT cis-PT complex. A, epitope-tagged NgBR and hCIT were transiently expressed in Expi293 cells from bicistronic vector and purified as described in the text. SA, putative signal anchor; TM, putative transmembrane domain. B, representative Western blot monitoring purification of NgBR–hCIT complex. Ex, total crude extract, P, 200,000 × g membrane pellet; SUP, 200,000 × g supernatant after Triton X-100 solubilization of the membrane fraction; FT1 and FT2, flow-through from Strep-Tactin and nickel column, respectively; E1, elution from the Strep-Tactin; E2, final elution from the nickel column. C, Coomassie-stained SDS/PAGE showing SUP, FT1, E1, two (E2a), and eight times concentrated final fraction (E2b). D, size-exclusion chromatography profile of purified NgBR–hCIT cis-PT complex. UV absorption at 254 nm was measured as a readout for protein elution (left y axis, black line). Incorporation of ¹⁴C-labeled IPP into organic fraction was measured as a readout for cis-PT activity (right y axis, red line).