High-Throughput Expression PCR Used to Systematically Investigate Regulation of Translation Initiation in an E. coli Cell-Free Expression System
A number of publications describe strategies for optimization of heterologous protein expression in E. coli (an overview is given in Hannig and Makrides 1998). As the most important rate-limiting factor, the step of initiation of translation has been identified. Besides the start codon and a Shine-Dalgarno motive additional sequence characteristics seem to have an influence on the efficiency of translation initiation. Particularly important for translation initiation are sequence elements at the 5’-untranslated region of the mRNA. Makrides (1996) described different translational enhancer sequences such as, for example, a sequence from the T7-phage genel0 leader or a U-rich sequence from the 5 ’-untranslated region of some mRNAs like the atpE regulator from E. coli. The so-called downstream box, a sequence element of highly expressed T7 phage genes with homology to the ribosomal 16 S RNA, was also suggested as translational enhancer element (Sprengart et al. 1996). However, on the basis of this work, so far no universal expression vector could be proposed which is equally useful for every protein.
KeywordsGreen Fluorescent Protein Fluorescence Eukaryotic Gene Green Fluorescent Protein Gene Green Fluorescent Protein Fusion Initiation Region
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