Patent ID: 7419783
Filing Date: 2008-09-02
Classification: C07K,C12N

Abstract:
1. A method of identifying a leader peptide that directs protein export in bacteria, comprising the steps of: a) obtaining a library of nucleic acid sequences encoding mutated leader peptides; b) constructing a plurality of expression cassettes comprising said nucleic acid sequences encoding mutated leader peptides upstream of a nucleic acid sequence encoding a short-lived reporter protein, wherein said short-lived reporter protein is constructed by operably linking a cytoplasmic degradation sequence to the nucleic acid sequence encoding said reporter protein, wherein the short lived reporter protein is subject to degradation in the cytoplasm of bacteria; c) expressing said plurality of expression cassettes in bacteria; d) measuring expression of said reporter protein in said bacteria; and e) collecting bacteria cells having increased expression of said reporter protein relative to bacteria that do not express a leader peptide that directs protein export of said short lived reporter protein, wherein the mutated leader peptide expressed in said cells that have increased expression of said reporter protein is a leader peptide that directs export from the cytoplasm, whereby said export rescues said short-lived reporter protein from degradation in the cytoplasm.