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.Essentialstepsofthelysatepreparationweremodifiedandanalyzedwithrespect totheirimpacton in vitro proteinsynthesiscapacity,usingthegreenfluorescentprotein (GFP) as a target protein. Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional gel elec- trophoresis 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 com- parison among the different lysates. Conversely, depending on the activity profiles, up to7differentisoformsoftheelongationfactorEF-Tsweredetectedinthegelmaps. Keywords: Cell-free protein synthesis / Escherichia coli / Two-dimensional electrophoresis / Green fluorescent proteins / Elongation factor / Protein phosphorylation EL4023 Petra Theresia Schindler Sandra Baumann Matthias Reuss Martin Siemann Institut für Bioverfahrenstechnik, Universität Stuttgart, Stuttgart, Germany In vitro protein biosynthesis resting on cell-derived extracts is a suitable analytical tool to investigate prob- lems of cell-based protein expression, like toxicity or insolubility of target proteins in microorganisms [1]. More- over, this technique offers even more sophisticated appli- cations, such as the incorporation of unnatural amino acids [2], the production of functional monoclonal antibod- ies [3], or directed evolution based on ribosomal or poly- somal display, recently reviewed in [4]. In contrast to these remarkable innovations, the productivity of cell-free protein synthesis remained low, despite many attempts to detect the molecular causes for these limitations. A key requirement for future applications on a preparative scale is therefore to obtain a significant improvement of system performance. Herein, reasonably productive cell-free pro- tein synthesis systems have been derived from Escheri- chia coli (for a recent review see [4]), allowing an in vitro synthesis of up to 6 mg/mL of recombinant protein [5]. Bacterial cell-free systems are based on a crude cell extract (a so-called S30-lysate), containing all biocata- lysts necessary for protein expression (ribosomes, trans- lation factors, aminoacyl-tRNA synthetases, e.g.), and enzymesforenergyregeneration.Basedonaroutinepro- cedure published by Pratt [6], system performance in cul- tivation and some essential steps of the lysate prepara- tion were modified and analyzed with respect to their impact on in vitro protein synthesis capacity. In this con- text, variably manufactured lysates were examined by high resolution two-dimensional gel electrophoresis anal- ysis (HR-2-DE) to determine whether these preparative modifications caused substantial alterations in the protein composition of the final lysate. Thus, the presented stud- ies were essentially focused on the analysis of selected proteins directly involved in protein synthesis, namely translation factors and ribosomal proteins. For this pur- pose, different lysates with negligible, moderate, and high translational activity were produced and analytically compared. Cultivation: For the production of lysate A. Escherichia coli MSCC3010 was grown batchwise on LB complex mediumundercontrolledreactionconditionsina30Lbio- reactor. At the end of the exponential growth phase, cells were harvested with a cell concentration of 2.2 g/L. For the production of lysates B and C, the same strain was propagated batchwise on a mineral medium according to Wilms et al. [8]. Cells within the exponential growth phase were harvested at a concentration of 2.4 g/L and a maxi- mumspecificgrowthrateof0.8h ±1 . Lysate preparation: Lysate preparations were carried out as described by Pratt [6]. One of the most crucial steps within the described procedure is the so-called ªpreincu- bationº step, also called ªrun-off procedureº. The prepara- tion is carried out due to the generally believed necessity of releasing polysomes from endogenous mRNA [1, 4, 6] Correspondence: Dr. Martin Siemann, Institut für Bioverfah- renstechnik, Universität Stuttgart, Allmandring 31, D-70569 Stutt- gart, Germany E-mail: siemann@ibvt.uni-stuttgart.de Fax: +49-(0)711-685-5164 Abbreviation: GFP, green fluorescent protein 2606 Electrophoresis 2000, 21, 2606±2609  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/1313-2606 $17.50+.50/0