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
Life of this patent
Estimating the term…
Abstract
Novel DNA-binding proteins, especially repressors of gene expression, are obtained by variegation of genes encoding known binding protein and selection for proteins binding the desired target DNA sequence. A novel selection vector is used to reduce artifacts. Heterooligimeric 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.
Claims42
A cell culture comprising a plurality of cells, each cell bearing a selection vector, said selection vector comprising a first and a second operon, each comprising at leat one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, said cell culture being transformed with a variegated gene encoding potential DNA-binding proteins or polypeptides, where said cells collectively can express a plurality of different but sequence-related potential DNA-binding proteins or polypeptides.
A selection vector, said selection vector comprising a first and a second operon, each comprising at least one expressible gene, the genes of said first and second operons being different, a copy of the target DNA sequence being included in each operon and positioned therein so that under forward selection conditions the transformed cells enjoy a selective advantage if they express a protein or polypeptide which binds to said copies of the target DNA sequence, and further comprising a gene coding on expression for a potential DNA-binding protein or polypeptide, said gene comprising:
- (a) a coding region that codes on expression for a polypeptide, each domain of said polypeptide having at least 50% sequence identify to a known DNA-binding domain, and
- (b) a promoter operably linked to said coding region for controlling its expression.
Dependencies are inferred from the claim text.