Expression of functional eukaryotic proteins
AssigneeCalifornia Institute of Technology
InventorsFrances H. Arnold, Zhanglin Lin
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Abstract
This invention relates to the improved expression of evolved polynucleotide and polypeptide sequences encoding for eukaryotic enzymes, particularly peroxidase enzymes, in conventional or facile expression systems. Various methods for directed evolution of polynucleotide sequences can be used to obtain the improved sequences. The improved characteristics of the polypeptides or proteins generated in this maruer include improved folding, without formation of inclusion bodies, and retained functional activity. In a particular embodiment, the invention relates to improved expression of the horseradish peroxidase (HRP) gene and HRP enzymes. HRP mutants that are highly expressed, highly active, and/or thermostable, are disclosed.
Claims56
A method of obtaining and improving the production of a functional polypeptide by a host cell comprising the steps of:
- (a) providing at least one parent polynucleotide encoding a parent polypeptide,
- (b) altering the nucleotide sequence of the parent polynucleotide to produce a population of mutant polynucleotides;
- (c) transforming host cells with the mutant polynucleotides to express polypeptides;
- and
- (d) screening the polypeptides for a functional polypeptide having at least one modified property.
A method of obtaining and improving the production of a functional polypeptide by a host cell comprising the steps of:
- (a) providing an original parent polynucleotide encoding a parent polypeptide;
- (b) altering the nucleotide sequence of the parent polynucleotide to produce a population of mutant polynucleotides;
- (c) transforming host cells with the mutant polynucleotides to express polypeptides;
- (d) screening the polypeptides for a functional polypeptide having at least one modified property;
- (e) repeating steps (b)-(d) at least once to produce an additional generation of polypeptides, wherein a mutant polynucleotide encoding a functional polypeptide is employed as a new parent polynucleotide.
A polynucleotide encoding for a horseradish peroxidase which has one or more mutations at an amino acid position selected from 371, 131, and 223, and wherein the starting methionine residue is at position 0.
A polynucleotide encoding for a horseradish peroxidase which has at least one mutation selected from L371I and L131P.
A polynucleotide encoding for a horseradish peroxidase which has mutations at amino acids 131 and 223, and at least one additional mutation at at an amino acid position selected from 47, 93, 102, 226, 241, 303, wherein the starting methionine residue is at position 0.
A polynucleotide encoding for a horseradish peroxidase which has the amino acid mutations L131P land L223Q, and at leats one additional mutation selected from N47S, R93L, T102A, P226Q, K241T, and V303E.
A mutant horseradish peroxidase having at least one mutation selected from L131P and L223Q, N47S, R93L, T102A, P226Q, K241T, and V303E.
Dependencies are inferred from the claim text.