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US 5,512,434 AGranted
Expression cloning of a human phosphatase
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorsStuart A. Aaronson, Donald P. Bottaro, Toshio Ishibashi, Toru Miki
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Abstract
A method for the identification of related polypeptides, using an function-based selection criterion, is disclosed. A novel human phosphatase, and nucleotide sequences coding therefor, identified by the aforementioned method and designated VHR, is also disclosed.
Claims11
A method for identifying a DNA encoding a protein phosphatase, comprising
- (a) providing an autophosphorylating protein tyrosine kinase,
- (b) transfecting or infecting a bacterial host with a phagemid comprising
- (i) an origin of replication;
- (ii) a DNA test molecule;
- and
- (iii) a regulatory region which results in the expression of a test protein encoded by said DNA test molecule,
- (c) culturing the transfected or infected bacterial host in the presence of the phosphorylated form of said autophosphorylating protein tyrosine kinase such that phagemid-induced plaques are formed and said autophosphorylating protein tyrosine kinase and said test protein are brought in contact with each other;
- (d) screening the resulting phagemid plaques to identify plaques that contain the unphosphorylated form of said autophosphorylating protein tyrosine kinase;
- and
- (e) isolating phagemid DNA from plaques which contain the unphosphorylated form of said autophosphorylating protein tyrosine kinase, whereby said isolated-phagemid DNA encodes said protein phosphatase.
7 more elements
A method for identifying a DNA encoding a protein phosphatase, comprising
- (a) providing an autophosphorylating protein tyrosine kinase,
- (b) transforming a bacterial host with a plasmid comprising
- (i) an origin of replication;
- (ii) a selectable marker;
- (iii) a DNA test molecule;
- and
- (iv) a regulatory region which results in the expression of a test protein encoded by said DNA test molecule,
- (c) culturing the transformed bacterial host, in the presence of the phosphorylated form of said autophosphorylating protein tyrosine kinase, under selective conditions, such that plasmid-containing colonies are formed;
- (d) treating said colonies to bring said autophosphorylating protein tyrosine kinase and said test protein in contact with each other;
- (e) screening said colonies to identify colonies that contain the unphosphorylated form of said autophosphorylating protein tyrosine kinase;
- and
- (f) isolating plasmid DNA from colonies which contain the unphosphorylated form of said autophosphorylating protein tyrosine kinase, whereby said isolated-plasmid DNA encodes said protein phosphatase.
9 more elements
Dependencies are inferred from the claim text.