Novel buffer systems for macromolecular crystallization

Abstract

In protein crystallization, screening is initially performed to obtain an indication of the conditions under which a macromolecule might crystallize. These preliminary conditions are then optimized to produce (in a perfect world) well diffracting crystals; this process of optimization often involves fine grid screening around the initial conditions. An issue in optimization is to find factors which are independent, so as to simplify the analysis of the results of optimization trials. This is necessarily difficult with buffers, as a buffer and its pH range tend to be very highly correlated. Multi-buffer systems for pH modulation are presented which enable a broad pH range to be sampled without changing the chemical composition of the buffering component.

Crystallization of an apo form of human arginase: using all the tools in the toolbox simultaneously

“…The protein was concentrated to 53 mg ml À1 in HBS + MnCl 2 and further 'matrix-seeding' screening experiments were set up in the same manner: these screens again focused on mid-weight PEG-rich screens and protein with and without added ornithine. Hits consisting of thin multiple plates were observed in conditions containing 18-22% PEG MME 2000 and either malate-MES-Tris (MMT; Newman, 2004) buffer pH 7 or bistris buffer pH 6.5. These experiments were repeated with a second batch of protein that had been concentrated to 33 mg ml À1 .…”

A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo

“…In brief, the crystals were grown using the sitting drop vapor diffusion technique at 293°K with reservoir conditions of 118 mmol/L CaCl 2 , 18.5% (w/v) PEG 6000, and 10% (v/v) propionate-cacodylate-Bis-Tris propane buffer (pH 5.3; ref. 30). The crystal was harvested and cryoprotected by briefly soaking in a solution of the mother liquor with 20% of a 1:1 ethylene glycol/glycerol mixture added and flash cooled in the nitrogen stream at the MX-1 beamline of the Australian Synchrotron.…”

Selective on-line detection of boronic acids and derivatives in high-performance liquid chromatography eluates by post-column reaction with alizarin

“…To improve the composition of the alizarin solution, the effect of pH on the fluorescence intensity of alizarin/PBA mixtures was studied (a similar experiment has been published, where ARS was used instead of alizarin [14]), using an extended-range citrate/HEPES/CHES buffer system [15]. The largest difference in fluorescence intensity (=the highest sensitivity) between alizarin only and alizarin/PBA was obtained at pH values between 7 and 8 ( Fig.…”