Walk-through mutagenesis
OwnerRoberto Creainventor
Life of this patent
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Abstract
A method of mutagenesis by which a predetermined amino acid is introduced into each and every position of a selected set of positions in a preselected region (or several different regions) of a protein to produce library of mutants. The method is based on the premise that certain amino acids play crucial role in the structure and fuction of proteins. Libraries can be generated which contain a high proportion of the desired mutants and are of reasonable size for screening. This libraries can be used to study the role of specific amino acids in protein structure and function and to develop new or improved proteins and polypeptides such as enzymes, antibodies, single chain antibodies and catalytic antibodies.
Claims42
A method of mutagenesis of a protein, comprising introducing a predetermined amino acid into each of a set of selected sequence positions in a predefined region of the protein to produce a protein library comprising mutant proteins in which the predetermined amino acid appears at least once in essentially all of the selected sequence positions in the region.
A method of mutagenesis of a protein, comprising introducing one or more predetermined amino acids into each of a set of selected sequence positions in one or more predefined regions of the protein to produce a protein library comprising mutant proteins in which the predetermined amino acid appears at least once in essentially all of the selected sequence positions in the region.
A method of mutagenesis of a protein, comprising introducing a predetermined amino acid in each sequence position in a preselected region of the protein to produce a protein library comprising mutant proteins in which the predetermined amino acid appears at least once in essentially all positions in the region.
A method of mutagenesis, comprising;
- a. selecting a defined region of the amino acid sequence of the protein to be mutagenized;
- b. determining an amino acid residue to be inserted into amino acid positions in the defined region;
- c. synthesizing a mixture of oligonucleotides, comprising a nucleotide sequence for the defined region, wherein each oligonucleotide contains, at each sequence position in the defined region, either the nucleotide required for synthesis of the protein to be mutagenized or a nucleotide required for a codon of the predetermined amino acid, the mixture containing all possible variant oligonucleotides according to this criterion;
- and
- d. generating an expression library of clones containing said oligonucleotides.
A method of producing a mutant protein having a desired structure or function by walk-through mutagenesis, comprising;
- a. selecting a defined region of the amino acid sequence of the protein to be mutagenized;
- b. determining an amino acid residue to be inserted into amino acid positions in the defined region;
- c. synthesizing a mixture of oligonucleotides, comprising a nucleotide sequence for the defined region, wherein each oligonucleotide contains, at each sequence position in the defined region, either the nucleotide required for synthesis of the protein to be mutagenized or a nucleotide required for a codon of the predetermined amino acid, the mixture containing all possible variant oligonucleotides according to this criterion;
- d. generating an expression library of clones containing said oligonucleotides;
- e. screening the library to detect a clone encoding a mutant protein having the desired structure or function;
- and
- f. expressing a mutant protein having the desired structure or function by virtue of the presence of the oligonucleotide present in the clone detected in step (e).
A library of mutants of a protein, comprising mutant proteins in which a predetermined amino acid appears at least once in essentially every position in a region of the protein, wherein mutants containing at least one residue of the predetermined amino acid in a region of the protein comprise a proportion ranging from about 12.5% to 100% of the total number of different mutants in the library.
A library of HIV protease mutants, comprising mutant proteins in which three predetermined amino acids appear at least once in all positions of the active site region of the protease.
A method of producing a mixture of oligonucleotides for mutagenesis of a nucleotide sequence encoding a selected region of a protein to introduce a predetermined amino acid at each position in the region, comprising synthesizing a mixture of oligonucleotides comprising the nucleotide sequence for the preselected region, wherein each oligonucleotide contains, at each sequence position in the selected region, either a nucleotide required for synthesis of the amino acid of the region or a nucleotide required for a codon of the predetermined amino acid, the resulting mixture containing all possible variant oligonucleotides containing either of the two nucleotides at each position.
An instrument for DNA synthesis having ten reagent vessels, each of four vessels containing a different one of the four nucleotide synthons corresponding to the four nucleotides of DNA and each of six containing vessels containing one of the six different mixtures of two synthons.
Dependencies are inferred from the claim text.