Labeled protein and its producing method, labeling compound to be used in the method, and method for analyzing function of genes
View Patent ↗A protein having a labeling compound attached to its C-terminal, in which the compound comprises a label portion comprising a label substance and an acceptor portion comprising a compound having an ability of binding to a C-terminal of a synthesized protein when protein synthesis is carried out in a cell-free protein synthesis system or in a living cell is provided. Also, a method for producing the protein comprising the step of performing synthesis of a protein in a cell-free protein synthesis system or in a living cell in the presence of a labeling compound comprising a label portion comprising a label substance and an acceptor portion comprising a compound having an ability of binding to a C-terminal of a synthesized protein when protein synthesis is carried out in the cell-free protein synthesis system or in the living cell, the labeling compound being present at a concentration effective for the labeling compound to bind to the C-terminal of the synthesized protein is provided. Also, the labeling compound and a method for analyzing a function of a gene are further provided.
1. A method for analyzing a function of a protein, comprising the steps of:
i) producing a labeled protein which consists of a protein portion and a labeling compound chemically linked to a C-terminal of the protein portion by the steps of adding a nucleic acid containing a gene to a cell-free protein synthesis system as a template, and performing protein synthesis in the presence of the labeling compound to produce the labeled protein consisting of the protein portion and the labeling compound chemically linked to the C-terminal of the protein portion, wherein the labeling compound consists of a) an acceptor portion and b) a non-radioactive label substance chemically linked to the acceptor portion, wherein said acceptor portion comprises one member selected from the group consisting of puromycin, a puromycin derivative having the ability to bind to the C-terminal of a synthesized protein when protein synthesis is performed in a cell-free protein synthesis system or in a living cell, 3′-N-aminoacylpuromycin aminonucleoside and 3′-N-aminoacyladenine aminonucleoside, wherein said labeling compound is present at a concentration effective for said labeling compound to bind to the C-terminal of the synthesized protein, said protein being translated in the presence of said labeling compound;
ii) contacting the labeled protein with a target molecule; and
iii) determining an interaction between the labeled protein and the target molecule, to thereby analyze a function of the labeled protein.
2. The method according to claim 1 , wherein said acceptor portion comprises puromycin.
3. The method according to claim 1 , wherein said acceptor portion comprises a puromycin derivative.
4. The method according to claim 3 , wherein said puromycin derivative is one member selected from the group consisting of ribocytidyl puromycin, deoxycytidyl puromycin and deoxyuridyl puromycin.
5. The method according to claim 1 , wherein said non-radioactive label substance is a fluorescent dye.
6. The method according to claim 5 , wherein said fluorescent dye is fluorescein.
7. The method according to claim 1 , wherein said labeling compound is fluoresceinylphosphopuromycin or fluoresceinylthiophosphopuromycin.