Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis : Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis

Abstract: Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.