Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation

The evaluation of the translation capacity of cells that produce recombinant proteins can be made by monitoring their ribosomal composition. In a previous use of asymmetrical flow field-flow fractionation (AsFIFFF) for this purpose the overall analysis time was more than 1 h and 40 min, based on a standard protocol for cell harvest, washing, cell disruption, and the final 8-min AsFIFFF determination of ribosome and subunits. In the present work the overall analysis time was reduced to 16 min. The washing step was deleted and a time-consuming freeze-thaw cycle. Cell disruption was obtained by a time-minimized lysozyme and detergent treatment for 1.5 min, respectively. The ribosomal material was finally fractionated and quantified in only 6 min, without previous centrifugation, using AsFIFFF. The great time reduction will enable the future use of AsFIFFF at-line to a growing cell cultivation, continuously monitoring the change in ribosomal composition or in other applications requiring high sample throughput. To demonstrate the high efficiency of the method the ribosome and tRNA composition in an Escherichia coli cultivation was monitored every half an hour, giving 18 measurements across the complete growth curve, a frequency of data enough to make decisions about induction or termination of the cultivation. (C) 2003 Elsevier Science (USA). All rights reserved.