Method for creating optimized regulatory regions affecting protein expression and protein trafficking

摘要

This invention discloses a method for optimizing the production of a polypeptide in a cell comprising the steps of identifying a regulatory region within a nucleic acid sequence to be mutagenized; preparing a nucleic acid vector comprising that region and encoding at least one polypeptide regulated by said regulatory region; deleting that region from the vector; producing a pool of random oligonucleotides; using PCR to introduce one random oligonucleotide into the position previously occupied by the regulatory region into each of a plurality of vectors to generate a pool of mutagenized vectors; introducing the mutagenized vectors into a cell sample; assaying for the expression of the polypeptide in that sample; and selecting and isolating those cells exhibiting optimized polypeptide expression. The regulatory regions contemplated within the scope of this invention include signal and protein trafficking sequences, ribosome binding sites, promoters, and translational regulatory sequences.