Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

Lee, H.-H.; Cherni, I.; Yu, H.; Fromme, R.; Doran, J.D.; Grotjohann, I.; Mittman, M.; Basu, S.; Deb, A.; Dörner, K.; Aquila, A.; Barty, A.; Boutet, S.; Chapman, H.N.; Doak, R.B.; Hunter, M.S.; James, D.; Kirian, R.A.; Kupitz, C.; Lawrence, R.M.; Liu, H.; Nass, K.; Schlichting, I.; Schmidt, K.E.; Seibert, M.M.; Shoeman, R.L.; Spence, J.C.H.; Stellato, F.; Weierstall, U.; Williams, G.J.; Yoon, C.; Wang, D.; Zatsepin, N.A.; Hogue, B.G.; Matoba, N.; Fromme, P.; Mor, T.S.

doi:10.1107/S2052252514014900

Figure 4
Affinity chromatography purification of CTBMPR. Protein samples from various steps in the purification process were resolved next to molecular-weight markers (lane 1) by SDS–PAGE and the gel was stained with Coomassie Brilliant Blue (upper panel). The whole cell lysate (lane 2) was spun down and the aqueous fraction (lane 3) was discarded. Membrane proteins were extracted from the pellet with βDDM (lane 4) purified over an affinity chromatography column. The flowthrough was collected (lane 5) and the column was extensively washed as described in the text (lane 6, first wash fraction; lane 7, last wash fraction). Elution required a larger volume of imidazole elution buffer to elute most of the protein bound to the column (lanes 8–10) than expected based on previous results with CTB^GPGPMPR (Matoba et al., 2008

). Immunoblotting was performed on the same samples using monoclonal 2F5 antibodies (lower panel).

IUCrJ

Volume 1| Part 5| August 2014| Pages 305-317

ISSN: 2052-2525

doi:10.1107/S2052252514014900

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