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Simplifies extraction and purification methods, mechanical or chemical extraction no longer required resulting in lower levels of impurities, no mis-folding due to over expression.
About
What is the problem? Purification is one of the major costs in the biosynthesis of proteins. Current mechanical or chemical extraction of proteins expressed in E. coli results in high levels of impurities being extracted along with the target protein. Furthermore over expression into cytoplasm or secretion into the periplasm can result in mis-folding. Our new Solution? Host cell secretion pathways have been manipulated to express proteins in the extracellular environment. Benefits of the new technology Simplifies extraction and purification methods, mechanical or chemical extraction is no longer required resulting in lower levels of impurities, no mis-folding due to over expression in cytoplasm. Background Gram negative bacteria have a number of functionally independent host cell secretion pathways, which can potentially be manipulated to express proteins in the extracellular environment. One such secretion pathway utilises autotransporter (AT) proteins, which, unlike other secretary pathways, are encoded by a single polypeptide. With three distinct domains a limitation of this pathway has been the difficulty in cleaving the passenger domain from the β-domain which is attached to the cell surface. A new system has been developed which allows cleavage and release of proteins into the extracellular environment, utilising an AT transporter system that incorporates a self-cleaving domain – “the secretion unit” from a minimal fragment of the SPATE-class AT C-terminal β-domain. This secretion unit comprises an α helix; a linker and a β-barrel region of the β-domain of the AT polypeptide. A secretion unit of less than 300 amino acids has been determined which, importantly, does not have to include any amino acid sequence from the passenger domain in order to direct efficient secretion and release of the passenger domain into the extracellular environment. This technology has a number of potential significant advantages over current bacterial expression systems, as it removes the need for extraction techniques; reduces the diversity and quantity of process impurities; increases process robustness; speeds-up process development times; reduces development and manufacturing costs; and speeds up time-to-market for the protein.