ENGINEERING OF LEADER PEPTIDES FOR THE SECRETION OF RECOMBINANT PROTEINS IN BACTERIA

摘要

The present invention provides methods of isolating of leader peptides capab le of directing export of heterologous proteins from the bacterial cytoplasm. T he methods rely on the screening of libraries of putative leader peptides or of leader peptide mutants for sequences that allow rapid export and thus can rescue a short-lived reporter protein from degradation in the cytoplasm. The mutant leader peptides identified herein are shown to confer significantly higher steady state levels of export not only for short-lived reporter prote in but also for other stable, long-lived proteins. These leader peptides can be used to direct or enhance protein secretion. The present invention further discloses methods for the export of cytoplasmically folded protein via the T at pathway. Proteins having disulfide bonds are first folded within the cytopla sm in suitable oxidizing mutant strains. Such cytoplasmically pre-folded protei ns containing disulfide bonds are then exported via the Tat pathway.