LARGE-SCALE PREPARATION OF FULLY DEUTERATED CELL COMPONENTS - RIBOSOMES FROM ESCHERICHIA-COLI WITH HIGH BIOLOGICAL-ACTIVITY

Some applications of NMR and of neutron scattering require fully deute rated biological material which should be highly active and available in large quantities. These requirements are hardly compatible since fu ll deuteration is achieved easily only if cells are grown in minimal m edia. This condition used in standard batch fermentation results in bo th low yields and reduced activities of the biological mass. Here we r eport a method which combines the apparently incompatible requirements taking advantage of a recent observation according to which the appea rance of growth inhibiting extracellular products could be prevented. The method was applied for growing Escherichia coli cells, strain MRE6 00rif (resistance against high doses of rifampicin is used as selectio n marker) on partially deuterated media (76% and 84% D2O) with glucose as carbon source and on deuterated acetate and succinate with 100% D2 O when full deuteration was to be achieved. The essential point for pr eserving the log-phase character of the cells is that the cultivation is carried out at substrate limiting conditions thus keeping the growt h rate at low levels (for glucose the growth rate, mu less-than-or-equ al-to 0.35 h-1, for acetate/succinate mu less-than-or-equal-to 0.1 h-1 ) which avoids the accumulation of the substrate or of by-products in the medium. Our data suggest that acetate is a main extracellular comp onent for accompanying or triggering the transition from logarithmic g rowth to stationary phase of E. coli cells cultivated on glucose as ca rbon source. The cells were first grown in fed-batch to high cell dens ities (above 50 g wet cells/l) under conditions of substrate limitatio ns. A steady-flow fermentation followed keeping the growth rate at abo ut mu of 0.1 h-1. Cells were harvested in kg quantities, the extracted ribosomes showed a normal complement of proteins, contained intact rR NA and were fully active. The ribosomal protein and rRNA fractions cou ld be efficiently reconstituted to highly active particles. In the cas e of full deuteration a matching point of 120% (tentative D2O scale) w as achieved. The reported method facilitates the preparation of deuter ated biological material for applications in NMR and neutron scatterin g analysis.