PROTEOLYTIC INACTIVATION OF SELECT PROTEINS IN BACTERIAL EXTRACTS FOR IMPROVED EXPRESSION

摘要

The present disclosure provides modified proteins that are capable of being cleaved by the protease OmpT1. The proteins can be modified in an exposed surface motif to incorporate OmpT1 cleavage sites. Also provided are nucleic acids encoding the modified proteins, bacterial cells that express the modified proteins, and cell free synthesis systems containing modified RF1. The disclosure further provides methods for reducing the deleterious activity of a modified protein in a cell free synthesis system by contacting the modified protein with OmpT1. Also provided are methods for reducing RF1 competition at an amber codon in the cell free synthesis system, and methods for expressing a protein in the cell free synthesis system. The modified proteins of the invention can be used to increase the yield of proteins having non-natural amino acids incorporated at an amber codon.

主权项

1. A functional Releasing Factor 1 protein (RF1) that is cleavable by an Outer Membrane Protein T1 (OmpT1), where a scissile OmpT1 peptide bond is located within the switch loop region corresponding to amino acids 287-304 of wild type RF1 (SEQ ID NO:1) and where the switch loop region is modified to include two adjacent basic amino acids that are positively charged at pH 7.0