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Optimisation of a transcription-translation coupled in vitro system


Iskakova,  Madina
Max Planck Society;

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Iskakova, M. (2005). Optimisation of a transcription-translation coupled in vitro system. PhD Thesis, Freie Universität, Berlin.

Cell-free protein synthesis exploits the catalytic machinery of the cell to produce active proteins. An in vitro system is flexible and well controlled, and it offers several advantages over conventional in vivo technologies such as easy ways for purification, synthesis of regulatory and/or toxic proteins, incorporation of artificial or modified amino acids that might be doted with isotopes required for NMR. Here I describe experiments exploring optimisation possibilities concerning yield and quality of the synthesised protein. Some experimental strategies also include expression of eukaryotic genes in prokaryotic expression systems. The following results have been achieved: 1: Quality criteria developed that allow a critical evaluation of parameters important for the coupled transcription/translation system or improving the yield and quality of the synthesized protein exploiting the features of the green fluorescent protein GFP. 2: The standard transcriptase used in overexpression studies in vivo and in vitro is the T7 polymerase. The fundamental difficulty with this enzyme is the fact that it is about six times faster than the E. coli transcriptase and thus uncouples transcription from translation, a possible reason for the fact that in vitro systems usually produce proteins with an activity of 30 to 60% only. We tested some slow mutants of T7 polymerase that approached the rate of the E. coli transcriptase and observed indeed a significant improvement up to 100% of the active fraction, although at the cost of lower yields. 3: A similar improvement of the active fraction was observed at lower incubation temperatures down to 20°C, again at the cost of lower yields. 4: According to literature data some amino acids are metabolised during in vitro incubations and thus could cause a limitation of protein synthesis. Indeed, we demonstrate that a second addition of amino acids in the middle of the incubation triggers a burst of further protein synthesis. Using this trick at 20°C pushed the yield of protein to almost that seen at 30°C, but now with an active fraction of 100%. In contrast, our analysis revealed that NTPs are not limiting the gene expression in vitro in our system (modified Roche RTS). 5: It is known that the codon usage of highly and lowly expressed proteins in E. coli differs dramatically. When we examined this point with human genes, to our surprise a corresponding difference could not be observed. Due to this fact it was possible to identify 11 tRNAs the corresponding codon are quite often used in human genes but rarely in E. coli genes. Therefore, for a good expression of eukaryotic genes in E. coli systems these 11 tRNAs should be added (and not only the 7 tRNAs supplied in systems from Novagen). 6: I outlined some ways to improve further the expression system.