Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
2001-05-08
2004-07-13
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S193000
Reexamination Certificate
active
06762041
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method for isotopically labeling a functional group of an amino acid residue of a protein and a protein whose amino acid residues have an isotopically labeled functional group.
As the genome analysis technique has been advanced, the genes of a variety of animals, plants and microorganisms have been revealed one after another. Moreover, efforts have recently been put into the post-genome studies, for example the development of pharmaceuticals based on the gene information. In particular, the structure of a protein encoded by specific genes obtained and the functions thereof have extensively been analyzed in order to elucidate the roles of the genes. This is because, if it is found that such a protein is, for instance, a receptor protein, one can find out agonists or antagonists by screening the ligands therefor. However, there has not yet been known any standardized method for analyzing functions and structures of proteins unlike the gene sequence analysis and accordingly, and it is necessary to use or select an appropriate method depending on the purposes. Among these, a method for labeling a target protein has widely been used because the method makes the detection of the target protein easy.
As means for labeling protein, there may be listed, for instance, a method for incorporating isotopes such as
2
H,
13
C and
15
N into the protein; and a method for chemically modifying the protein with a fluorescent reagent or a chelating agent. The development of proteins labeled with stable isotopes such as
2
H,
13
C and
15
N can considerably extend the scope of NMR applications and the isotope-labeled proteins have widely been used as a means indispensable to the structural studies with NMR (see, for instance, Ikura, M. et al. Science, 1992, 256:632). In addition, such isotope-labeled proteins have frequently been used in the functional analysis while making the most use of the fact that they can be detected by several techniques such as NMR and MS. On the other hand, the chemical modification of a protein with a fluorescent reagent or a chelating agent is suited to the detection of trace samples and therefore, it has widely been used for determining the distribution of proteins and for quantitative analysis of the activity of the proteins.
As characteristic properties of the foregoing stable isotopes, there may be listed, for instance, any chemical property of a protein is not fundamentally changed through the labeling with such an isotope at all. Accordingly, stable isotopes have been put into practical use in the structural and functional analysis of proteins. In particular, in the structural analysis of proteins by means of the NMR technique, there have been known a method for reducing the number of signals of
1
H present in a protein by labeling the protein with
2
H to thus make the analysis thereof easy (a negative labeling method) and a method in which highly sensitive nuclides such as
13
C and
15
N are substituted for C and N, respectively, present in the protein to thus utilize the isotope-labeled protein in the multi-dimensional NMR technique (a positive labeling method). At the present time, most of proteins having a molecular weight of not less than 6000 have been subjected to structural analysis using either of these methods.
Labeling a protein with a stable isotope is mainly conducted by constructing an expression system of
E.coli
and then cultivating
E.coli
in a culture medium containing a labeling compound. In this respect, examples of such labeling compounds include
13
C glucose,
15
NH
4
Cl,
13
C glycerol and a variety of labeling amino acids (see, for instance, Kainosho, M. et al. Biochemistry, 1982, 21:6273; and Ikura, M. et al. Biochemistry, 1990, 29:4659). There has also been reported a method for preparing a labeled protein by the use of an expression system such as yeast or animal cells or cell-free systems, in addition to
E.coli
system (Kigawa, T. et al. J. Biomol. NMR, 1995, 2:129). In these methods, a labeling compound is incorporated therein during the biosynthesis of peptide chains. Therefore, a protein may site-directively or uniformly be labeled. However, it is necessary to construct an expression system for individual proteins and this requires a great deal of labor and time. In case of peptides and proteins having a short chain length, they may be synthesized using labeled amino acids, but this method is limited in objects to which the method is applied because of the restriction in the chain length.
On the other hand, the chemical modification permits the direct labeling of any protein prepared without any pre-treatment and there are not so many restrictions. Moreover, there have been known chemically modifying agents suited to individual functional group-carrying residue such as glutamic acid residue, aspartic acid residue and histidine residue, respectively and therefore, any protein can be labeled irrespective of the composition of the peptide chain thereof. However, the chemical modification may, for instance, add a functional group having characteristic properties different from that of a natural protein. Accordingly, some of the functions and/or the structure of the natural protein may sometimes be damaged and there is a limit in the application of the chemical modification to the functional analysis and the structural analysis.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for isotopically labeling a functional group of an amino acid residue of a protein.
It is another object of the present invention to provide a protein whose amino acid residue has a functional group isotopically labeled.
The use of such a protein whose amino acid residue has a functional group isotopically labeled would permit the functional analysis and the structural analysis of the isotopically labeled protein.
The inventors of this invention have conducted various studies to solve the foregoing problems associated with the conventional techniques, have found that the functional group possessed by an amino acid residue of a protein can be labeled with an isotope while making use of the activity of an enzyme and have thus completed the present invention.
Accordingly, the present invention relates to a method for isotopically labeling a protein, which comprises the step of bringing the protein to be labeled, an isotope-labeling compound and an enzyme into contact with one another. More specifically, the present invention pertains to a method for isotopically labeling a protein, which comprises the step of bringing the protein to be labeled, an isotope-labeling compound and an enzyme into contact with one another to thus replace a desired functional group possessed by an amino acid residue of the protein with an isotope-labeling group derived from the foregoing isotope-labeling compound through the action of the enzyme.
In particular, the present invention relates to a method for isotopically labeling a protein, which comprises the step of acting a transglutaminase on the glutamine residue of a protein to label the carboxyamide nitrogen of the glutamine residue.
In addition, the present invention also embraces a protein isotopically labeled according to such a method.
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Boyd et al., “The Influence of a Scaler-Coupled Deuterium Upon the Relaxation of a N15 Nucleus and its Possible Exploitation as a Probe of Side-Chain Interactions in Proteins” (1997) J. Biol. Chem., 124(1), 61-71.*
Signorini et al., “Identification of Transglutaminase Activity in the Leaves of Silver Beet (Beta vulgarisL.)” (1991) Journal of Plant Physiology, 137(5), 547-52, in Database CAPLUS, DN 114:244282.*
Takagi et al., “Modification of Transglutaminase A
Shimba Nobuhisa
Suzuki Ei-ichiro
Yokoyama Keiichi
Ajinomoto Co. Inc.
Weber Jon P.
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