Extracellular secretion of proteins expressed in
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
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 ╬▓-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 ΓÇô ΓÇ£the secretion unitΓÇØ from a minimal fragment of the SPATE-class AT C-terminal ╬▓-domain. This secretion unit comprises an ╬▒ helix; a linker and a ╬▓-barrel region of the ╬▓-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.
Inventor: SEVASTSYANOVICH, Yanina Romanovna | LEYTON, Denisse | HENDERSON, Ian Robert
Priority Number: WO2012110821A1
IPC Current: C12N000952 | C07K0014195
Assignee Applicant: The University of Birmingham
Title: PROTEIN SECRETION | SÉCRÉTION DE PROTÉINE
Usefulness: PROTEIN SECRETION | SÉCRÉTION DE PROTÉINE
Summary: For secreting a polypeptide from periplasm (claimed).
Novelty: New expression construct for secreting polypeptide from periplasm comprises nucleic acid sequence encoding secretion unit peptide having specific amino acids of C-terminus of serine protease autotransporter of enterobacteriaceae polypeptide
生物醫學
醫藥成分
美國
