Abstract
Cell-free extracts (lysates) from Escherichia coli were used for protein
synthesis in vitro. Essential steps of the lysate preparation were
modified and analyzed with respect to their impact on in vitro protein
synthesis capacity, using the green fluorescent protein (GFP) as a
target protein. Variably manufactured lysates of low, medium and higher
protein synthesis activity, were examined by high resolution
two-dimensional get electrophoresis to determine whether the
modifications result in substantial alterations in protein composition
of the final lysate. The total number of proteins calculated from the
get maps did not vary for lysates with different activity and thus
cannot serve as an evaluation parameter. Ribosomal proteins RP-S1,
RP-L9, and RP-L10 were found in stoichiometric amounts for each of these
lysates and in equal concentrations in comparison among the different
lysates. Conversely, depending on the activity profiles, up to 7
different isoforms of the elongation factor EF-Ts were detected in the
gel maps.
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