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

Arfvidsson, Cecilia LU and Wahlund, Karl-Gustav LU (2003) In Analytical Biochemistry 313(1). p.76-85
Abstract
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 (AsFlFFF) 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 AsFlFFF 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... (More)
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 (AsFlFFF) 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 AsFlFFF 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 AsFlFFF. The great time reduction will enable the future use of AsFlFFF 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. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Analytical Biochemistry
volume
313
issue
1
pages
76 - 85
publisher
Elsevier
external identifiers
  • wos:000181093100011
  • pmid:12576061
  • scopus:0037331018
ISSN
1096-0309
DOI
language
English
LU publication?
yes
id
fa939e3c-fb04-4aa7-8274-ab7e56c8abc2 (old id 132721)
date added to LUP
2007-06-26 12:39:54
date last changed
2018-05-29 10:48:49
@article{fa939e3c-fb04-4aa7-8274-ab7e56c8abc2,
  abstract     = {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 (AsFlFFF) 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 AsFlFFF 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 AsFlFFF. The great time reduction will enable the future use of AsFlFFF 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.},
  author       = {Arfvidsson, Cecilia and Wahlund, Karl-Gustav},
  issn         = {1096-0309},
  language     = {eng},
  number       = {1},
  pages        = {76--85},
  publisher    = {Elsevier},
  series       = {Analytical Biochemistry},
  title        = {Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation.},
  url          = {http://dx.doi.org/},
  volume       = {313},
  year         = {2003},
}