Investigation of translation dynamics under cell-free protein biosynthesis conditions using high-resolution two-dimensional gel electrophoresis.
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.
Schindler
P T
PT
Institut für Bioverfahrenstechnik, Universität Stuttgart, Germany.
Macherhammer
F
F
Arnold
S
S
Reuss
M
M
Siemann
M
M
D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D015203:Reproducibility of Results
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins