Abstract
The intracellular alarmone guanosine 3 `,5 `-bis(diphosphate) (ppGpp)
has been thoroughly investigated over the past 40 years and has become
one of the best-known effectors in bacterial physiology. ppGpp is also
of great importance for biotechnological applications. Systems biology
research, involving experimental and mathematical approaches, has
contributed a great deal to uncovering the alarmone's complex regulatory
effects. HPLC analysis and UV detection are used to quantify
intracellular ppGpp. The samples analyzed also contain other
phosphorylated guanine nucleotides and, therefore, are spiked with a
standard ppGpp solution. A rapidly growing number of laboratories are
turning to synthesizing the nucleotide in vitro involving time-consuming
protocols and yielding only low amounts of ppGpp. The current article
provides a protocol for the preparation of large quantities of a
ribosome extract that contains high ppGpp synthesis activity. The
demonstrated upscaling from shaking flask to bioreactor cultivation
involves the continuous and refrigerated harvest of the biomass. C-13
NMR analysis enabled the structural characterization of the synthesis
product and was complemented by mass spectrometry and methods that are
commonly used to identify ppGpp. (C) 2008 Elsevier Inc. All rights
reserved.
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