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 2
(a) Separation of aggregates and monomers from the pentameric CTB^GPGPMPR protein by gel filtration on a Superdex 200 column. Assembly status was estimated from parallel resolution of molecular-mass standards (not shown). Inset, tryptophan fluorescence emission spectra of pentameric CTB^GPGPMPR in pooled gel-filtration fractions corresponding to the major peak in (a). 1 (green), pentamers; 2 (blue), concentrated (Centricon 100) pentamers. Excitation was at 280 nm. (b) Proteins in the unconcentrated metal-affinity chromatography (MAC) eluate and in the size-exclusion chromatography (SEC) fraction corresponding to the main peak of the chromatogram in (a) were resolved by SDS–PAGE under nondenaturing (ND; no DTT and no boiling) and denaturing (D) conditions. Molecular-weight standards indicate that CTB^AAAAMPR is organized into SDS-stable pentamers. The compact pentamers have a slightly higher electrophoretic mobility than expected based on their mass alone.

IUCrJ

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

ISSN: 2052-2525

doi:10.1107/S2052252514014900

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