The effects of filtration on protein nucleation in different growth media

Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease dependent on the membrane cut-off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results. The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time-dependent change in the size or the number of crystals per drop. Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time-dependent effect.