Generation and selection of novel DNA-binding proteins and polypeptides
AssigneeProtein Engineering Corporation
InventorsRobert C. Ladner, Sonia K. Guterman, Rachel B. Kent, Arthur C. Ley
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Abstract
Novel DNA-binding proteins, especially repressors of gene expression, are obtained by variegation of genes encoding known binding proteins and selection for proteins binding the desired target DNA sequence. A novel selection vector may be used to reduce artifacts. Heterooligomeric proteins which bind to a target DNA sequence which need not be palindromic are obtained by a variety of methods, e.g., variegation to obtain proteins binding symmetrized forms of the half-targets and heterodimerization to obtain a protein binding the entire asymmetric target.
Claims48
A method of obtaining first and second genes encoding first and second homooligomeric DNA binding proteins which hybridize to form a hybrid heteroologomeric DNA binding protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target-sequence and right target subsequence each of at least 4 base pairs length, said method comprising:
- producing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two essentially dyad-symmetric DNA-binding domains, where said first target sequence is a palindrome or gapped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, and where said second target sequence is a palindrome or gapped palindrome and comprises said right target subsequence and a palindrome-completing subsequence, whereby one of the DNA-binding domains of the second DNA-binding protein binds to the right target subsequence, said genes being produced by a process of at least partially random mutation followed by selection for the binding of the corresponding protein to the corresponding target sequence,
- wherein said first and second proteins can hybridize so as to obtain a heterooligomeric DNA-binding protein comprising a DNA-binding domain recognizing the left target subsequence and a DNA-binding domain recognizing the right target subsequence
- whereby said heterooligomeric protein has an affinity for the ultimate target DNA.
A method of obtaining genes encoding a heterooligomeric protein which binds to a predetermined ultimate target double stranded DNA sequence, said sequence being nonpalindromic, said sequence comprising a left target subsequence and a right target subsequence each of at least 4 base pairs lengths, said method comprising:
- (a) providing a first gene encoding a first DNA-binding oligomeric protein binding to a first target sequence and a second gene encoding a second DNA-binding oligomeric protein binding to a second target sequence, wherein said first and second DNA-binding proteins each have at least two dyad-symmetric DNA-binding domains, wherein said first and second DNA-binding proteins each have a dimerization interface, where said first target sequence is a palindrome or gaped palindrome and comprises said left target subsequence and a palindrome-completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the first DNA-binding protein binds to said left target subsequence, and where said second target sequence is a palindrome or gaped palindrome and comprises said right target subsequence and a palindrome completing subsequence, whereby one of the dyad-symmetric DNA-binding domains of the second DNA-binding protein binds to the right target subsequence,
- (b) variegating the dimerization interface of the protein encoded by one of said first or second genes to obtain variegants thereof and reverse selecting for expression from said variegant of a first oligomerization mutant protein, encoded by a variegant of said variegated gene, which is no longer capable of forming a homooligomer that can bind to said first or second target sequence, respectively, and verifying that said oligomerization mutant protein maintains a tertiary structure similar to the protein form which is descended,
- (c) variegating the dimerization interface of the protein encoded by the other of said first or second genes to obtain variegants thereof,
- (d) providing host cells carrying the gene encoding said first oligomerization mutant protein and a variegant gene of step (c), each operably linked to a promoter functional in the host cell, and
- (e) forward selecting for expression from a step (c) variegant gene of a second oligomerization mutant protein which is capable of forming a heterooligomer with said first oligomerization mutant protein, said heterooligomer binding said ultimate target DNA sequence, and
- (f) isolating the genes encoding said heterooligomer.
Dependencies are inferred from the claim text.