Zusammenfassung
A cell-free extract from Escherichia coli, generated through a routine
procedure according to Chen and Zubay (Methods Enzymol. 1983, 101,
674-690), was used for an in vitro protein synthesis. High-resolution
two-dimensional gel electrophoresis (2-DE) was exploited to investigate
the protein composition of the cell-extract and its dynamic development
during a 24 h-period of cell-free protein synthesis performed in a
membrane reactor device. Green fluorescent protein (GFP) was chosen as a
target protein to be produced in a cell-free reactor because of its
functional activity, which can easily be monitored by measurement of
fluorescence, and because of its high sensitivity. GFP synthesis was
observed by a standard fluorescence assay and was correlated to a
quantitative assessment of the silver-stained GFP spot appearing on 2-DE
gel maps. A constant protein synthesis rate was obtained for at least 8
h of process operation. While declining continuously, protein synthesis
stopped entirely after 24 h. Both, the total protein content and total
number of detectable spots were found to decrease over the reaction
time, due to proteolytic digestion and protein precipitation. Certain
proteins taking part in the translation process, such as the elongation
factors (EF-Tu, EF-Ts) and the ribosomal protein RP-L9, were identified
by Edman N-terminal sequencing and have thus been considered for
reaction evaluation. The dynamics obtained during the entire process
suggest that these translational factors were likewise affected by
proteolytic decay.
Nutzer
