Rational library
AssigneeUni'TargetingResearch AS
InventorsBeate Stern, Ian Fraser Pryme, Hanne Ravneberg
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Abstract
The invention relates to a method to generate rational libraries comprising genetic elements which are involved in transcriptional and/or translational regulation of a gene and devised to increase the production yield of the encoded protein as well as to the rational library and to the application of said rational library.
Claims21
A method to generate rational libraries comprising genetic elements which are involved in the expression of a gene and devised to increase the production yield of the encoded protein, comprising the steps of
- a) providing a genetic element to be optimised for expression capacity and defining at most 18 nucleotide residues, either non-coding or coding for at most 6 amino acid residues at specific positions in said genetic element to be randomised,
- b) amplifying said genetic element, said genetic element being part of a double stranded DNA plasmid being a preliminary vector or a final vector and subjecting said genetic element to randomisation and generating a pool of genetic element variants,
- c) amplifying said pool of genetic element variants being part either of a preliminary vector, thus generating a pre-made library or being part of a final vector, thus generating a final library, or
- d) introducing said pool of genetic element variants being part of a preliminary vector into a recipient vector in a seamless manner, thus generating a final library,
- e) transforming said final library into eukaryotic cells and
- f) obtaining a eukaryotic cell pool containing a rational library comprising up to 418≈6.9×1010 different vector variants.
A method to identify a clonal cell line harbouring a vector variant where said clonal cell line produces a protein of interest at the highest amount comprising the steps of
- a) generating the genetic element variants in a vector containing the gene encoding the protein of interest or incorporating said genetic element variants from a pre-made library into a vector containing the gene encoding the protein of interest according to any of preceding claims,
- b) screening for the cell clone that produces the protein of interest to the highest level and
- c) obtaining a clonal cell line from the rational library that mediates the highest level of production of the encoded protein.
A rational library based on a vector containing different genetic elements which have been seamlessly cloned, said rational library containing up to 7×1010 different vector variants wherein each variant contains at most 18 changed nucleotides, either non-coding or coding for at most 6 amino acid residues, at specific positions in one of the genetic elements and wherein each vector variant mediates a different expression level of the encoded protein of interest as compared to the non-modified vector.
Dependencies are inferred from the claim text.