Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1998-11-13
2001-05-08
Prouty, Rebecca E. (Department: 1652)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S350000, C530S300000, C536S026300, C536S026600
Reexamination Certificate
active
06228994
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a labeled protein and its producing method and a labeling compound to be used in the method. Also the present invention relates to a method for analyzing function of genes.
For labeling of protein expressed in cell-free translation systems and living cells is used generally a radiolabeling method that involves incorporating an amino acid labeled with a radioactive element such as
35
S,
3
H,
14
C or the like into the translation product. However, amino acids may be used in reactions other than protein synthesis so that it happens that substances other than the translation product may be also labeled. On the other hand, the labeling method specific to the translation product includes the following method. To the &egr;-amino group of lysine, biotin is covalently linked, and the product is further linked through an ester linkage to tRNA having an anticodon for lysine to synthesize a complex (biotin-lysine-tRNA), which is put into a cell-free translation system to biotinate the translation product. The translation product is electrophoresed and then transferred on a membrane, and allowed to chemiluminesce with an alkali phosphatase by using a fusion protein of the alkaline phosphatase and streptoavidine. This chemiluminescence is recorded using X-ray film or the like for identifying the translation product (Promega, (1993) Technical Bulletin, No. 182, p.2). However, this method suffers from extreme instability of the synthesized biotin-lysine-tRNA (for 6 months at −70° C.) and is expensive. Further, there is the problem that the procedure for identification is complicated and time-consuming. This is disadvantageous in automation for processing on a large scale. The translated protein is modified by biotin at a plurality of lysine side chains so that there is the possibility that its function and structure vary from the original one.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a methodology which satisfies conditions such as 1) simplicity, 2) economically competitiveness, 3) long-term stability, and 4) no adverse influence on the function and structure of the translation product in labeling of translated protein in a cell-free translation system and living cells.
As a result of intensive investigation with view to overcoming the above-described problems, it has now been found that puromycin or its derivatives binds or bind to the C-terminal of a translated protein in a final concentration of 10 to 0.01 &mgr;M. It has also been found that the compound obtained by chemically linking a fluorescent substance such as fluorescein to puromycin binds to the C-terminal of the translated protein like puromycin and use of this enables one to identify proteins. That is, by adding the fluorescent puromycin to a cell-free translation system to carry out reaction, and then subjecting the product to gel electrophoresis, the gel without further processing can be read by a fluorescent image analyzer to identify the translated protein without difficulty. The present invention has been completed based on these findings.
Thus the present invention provides a labeling compound for labeling a protein, which 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, and a protein having the labeling compound attached to its C-terminal.
According to another aspect of the present invention, there is provided a method for producing the protein comprising the step of carrying out 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.
Further, according to another aspect of the present invention, there is provided a method for analyzing a function of a gene, comprising the steps of: adding a nucleic acid containing the gene to a cell-free protein synthesis system as a template; carrying out protein synthesis in the presence of a labeling compound to obtain a protein having the labeling compound attached to the C-terminal of the protein, the labeling compound being present at a concentration effective for the labeling compound to bind to the C-terminal of the synthesized protein; and analyzing a function of the labeled protein.
The label portion preferably comprises a radioactive substance or a non-radioactive label substance.
The acceptor portion preferably comprises a nucleic acid derivative. Alternatively, the acceptor portion preferably comprises a compound comprising a nucleic acid and an amino acid or an amino acid derivative which are bound to each other. More preferably, the acceptor portion comprises a compound comprising 2′- or 3′-aminoadenosine or its derivative and an amino acid or an amino acid derivative which are bound to each other. Particularly preferably, the acceptor portion comprises puromycin or its derivative.
The analysis of the function of protein preferably comprises determination of a protein-protein interaction, determination of a protein-nucleic acid interaction, or determination of an interaction between a protein and a ligand capable of specifically binding to the protein.
The labeling compound of the present invention is useful in detection and identification of a protein that is expressed in various cell-free protein synthesis systems or in living cells. In the future, the identification of corresponding proteins is most important theme in the functional analysis of genes that accumulate by genome analysis. The present invention provides effective means for increasing the efficiency of or automating analysis of function of protein such as a protein-nucleic acid interaction or a protein—protein interaction.
REFERENCES:
patent: 0 962 527 (1999-12-01), None
patent: 98/16636 (1998-04-01), None
Progress in Biophysics and Molecular Biology, XIIth International Biophysics Congress, abstract No. p-A5-04 (Aug. 1996).
Promega Technical Bulletin, No. 182 (Sep. 1993).
N. Nemoto et al.,FEBS Letters, 414, 405-408 (1997).
“Program and Proceedings of the 20thAnnual Meeting of the Molecular Biology Society of Japan”, Nov. 15, 1997, pp. 121, 546 and 547, together with a summarized translation.
Miyamoto Etsuko
Nemoto Naoto
Yanagawa Hiroshi
Mitsubishi Chemical Corporation
Prouty Rebecca E.
Wenderoth , Lind & Ponack, L.L.P.
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