In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity.
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 gel 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 gel 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.
Schindler
P T
PT
Institut für Bioverfahrenstechnik, Universität Stuttgart, Germany.
Baumann
S
S
Reuss
M
M
Siemann
M
M
D001426:Bacterial Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D019149:Bioreactors; D002457:Cell Extracts / Q000302:isolation & purification; D002474:Cell-Free System; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis; D009994:Osmolar Concentration; D010445:Peptide Elongation Factors / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D015203:Reproducibility of Results; D012269:Ribosomal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D013048:Specimen Handling / Q000379:methods; D013696:Temperature; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D002457:Cell Extracts; D003470:Culture Media; D008164:Luminescent Proteins; D010445:Peptide Elongation Factors; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C036506:elongation factor T; D049452:Green Fluorescent Proteins