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bc04-09-20.9.rz 24.09.2001 16:14 Uhr Seite 22 Rapid Translation System RTS 500 E. coli HY Kit – the Second Generation Cell-free System Expressing Milligram Amounts of Protein Erhard Fernholz*, Manfred Watzele, Claudia Kirr, and Bernd Buchberger Roche Molecular Biochemicals, Penzberg/Germany *corresponding author: [email protected] Cell-free protein expression has only been used as an analytical tool because of the low protein yields that could be obtained. With the Rapid Translation System RTS 500 HY it is now possible to express milligram amounts of protein in less than 24 hours. It also enables researchers to label proteins with high specificity and cost-effectiveness for X-ray and NMR analysis. Introduction RTS After the completion of the human genome project, numerous structural genomics initiatives were launched to promote the basic understanding on how nature works at the protein level. For this purpose, there is a demand for rapid protein expression systems which guarantee high yields and are convenient and amenable to automation. Cell-free protein expression has a strong potential to meet these requirements because handling of intact cells (e.g., transformation, fermentation, lysis) is not necessary. In fact, this methodology only involves liquid handling and can thus be adapted to automated processes. Low yield has been the limiting factor for many years in cell-free protein expression making this method suitable only as an analytical tool. The development of the RTS 500 E. coli Circular Template Kit in the year 2000 has marked the beginning of preparative scale, cell-free protein expression. The newly developed RTS 500 E. coli HY Kit now enables expression of up to 5 mg protein/ml within a day. This kit uses the Continuous Exchange Cell-Free (CECF) Table 1: Features of available RTS 100 and RTS 500 platforms Feature CECF-technology HY biochemistry Standard reaction volume RTS 500 Instrument Standard reaction time Protein yield 22 BIOCHEMICA · No. 4 · 2001 RTS 100 E. coli HY Kit RTS 500 E. coli HY Kit no yes 50 µl yes yes 1 ml RTS 500 E. coli Circular Template Kit yes no 1 ml not required 2 to 4 h required required 24 h 24 h up to 400 µg/ml up to 5 mg/ml up to 500 µg/ml technology and is based on the RTS 500 E. coli Circular Template Kit with improved reaction conditions (Table 1). Together with the recently introduced RTS 100 E. coli HY Kit for small scale protein expression reactions, this new kit will enable researchers to speed up their gene-to-protein experiments. Since the amino acids are now separated from the reaction and feeding mixtures, the design of the kit allows the incorporation of labeled amino acids. Materials and Methods The Rapid Translation Systems RTS 100 E. coli HY, RTS 500 E. coli Circular Template and RTS 500 E. coli HY were operated according to the pack insert. In case of Green Fluorescent Protein (GFP) expressions, the reaction chamber was half-filled to allow cotranslational oxidative maturation. In general, the synthesized protein was Reaction Chamber Stir Bar Membrane • DNA Template • T7 RNA Polymerase • Translation Machinery E. coli • Energy Regenerating System Inhibitory By-Products Supply • Amino Acids • Energy Substrates • Buffer Components • NTPs Feeding Chamber Stir Bar " Figure 1: Continous Exchange Cell Free (CECF) protein expression with the RTS 500. The RTS 500 device is composed of a reaction chamber, a semi-permeable membrane, and a feeding chamber. Transcription and translation take place simultaneously in the reaction chamber ROCHE MOLECULAR BIOCHEMICALS http://biochem.roche.com a b b a b a b a b a b β-Galactosidase (116 kD) Green Fluorescent Protein (27 kD) ErythropoetinThioredoxin Fusion Protein (33 kD) a b Erythropoetin (22 kD) a Seryl tRNA Synthetase (64 kD) Seite 23 CMV Small Capsid Protein 1049 (8.5 kD) b 16:14 Uhr Chloramphenicol Transferase (CAT) (22 kD) a Fc-γ-Receptor (30 kD) M.W. (kD) 24.09.2001 GFP-Parathormon Fusion Protein (36 kD) bc04-09-20.9.rz a b 98 52 31 19 17 11 6 3 # Figure 2: Comparison of the RTS 500 E. coli HY Kit and the RTS 500 E. coli Circular Template Kit. Proteins of different size and nature were expressed in the HY Kit (a) and the Circular Template Kit (b) GFP/Methionine (M = 149.2 g/mol) GFP/Selenomethionin (M = 196.1 g/mol) 28259.0 28491.0 28231.0 28464.0 Intensity (cps) For seleno-methionine labeling, the methionine in the standard protocol was replaced with 0.5 mM selenomethionine (final concentration). Results 28264.0 The new RTS 500 E. coli HY Kit is based on the CECF technology. As with the RTS 500 E. coli Circular Template Kit, the device at the center of this technology includes a 1-ml reaction chamber and 10-ml feeding chamber separated by a 10 -kD membrane. This set-up allows continuous replenishment of the critical components of the transcription/translation reaction (Figure 1). Protein expression proceeds for up to 24 hours, leading to substantially higher yields compared to methods that do not employ the CECF technology. Several changes in the biochemistry have led to a significant increase in yield making the high-yield version up to 10 times more productive than the RTS 500 E. coli Circular Template Kit (Figure 2). Because similar reaction conditions are also used in the RTS 100 E. coli HY Kit, the two platforms can be used together. The scalability allows economical screening of conditions (e.g., different sequence variations for the same gene) in the RTS 100 E. coli HY Kit with the possibility of obtaining enough protein for extended analysis using the same conditions in 24 hours with the RTS 500 E. coli HY Kit. Since the protein yield is sufficient for structural analysis, the incorporation of seleno-methionine was tested. Using the same concentration of seleno-methionine instead of ROCHE MOLECULAR BIOCHEMICALS http://biochem.roche.com RTS quantified via Coomassie-stained SDS-polyacrylamide gels and – if required – via an activity test (e.g., fluorescence for GFP) using isolated protein as a reference. 28591.0 " Figure 3: Mass spectroscopy analysis of seleno-methionine-labeled GFP. The shift in mass of 232 Da indicates the expected incorporation of 5 seleno-methionine residues (5x46.9 = 234.5) (Data kindly provided by N. Budisa and R. Huber, MPI for Biochemistry, Martinsried/Germany) methionine, all methionines could be readily replaced. This was verified by mass spectroscopy (Figure 3). Methionine is now separated from all other reagents in the kit and can therefore be easily replaced. Similarly, the exchange of amino acids for labeled amino acids to obtain protein for use in NMR spectroscopy was tested (see article by Ho S. Cho on page 27). For this purpose, all amino acids from the kit can be replaced with a tailored amino acid mixture, using the RTS Amino Acid Sampler and labeled amino acids of your choice. Product Pack Size Cat. No. RTS 500 Instrument 1 instrument 3 064 859 RTS 100 E. coli HY Kit 1 kit (24 reactions) 1 kit (96 reactions) 3 186 148 3 186 156 RTS 500 E. coli HY Kit 1 kit (2 reactions) 1 kit (5 reactions) 3 246 817 3 246 949 BIOCHEMICA · No. 4 · 2001 23