Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
1999-02-25
2003-01-14
Allen, Marianne P. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C536S023100, C514S002600, C530S300000
Reexamination Certificate
active
06507788
ABSTRACT:
BACKGROUND OF THE INVENTION
(i) Field of the Invention
The present invention relates to a method for identifying putative peptides from nucleotide or peptide sequences of unknown function such as both nucleic acid and peptide precursors of a peptide comprising an amidated C-terminal end and, more particularly, to a method wherein putative precursor peptides are identified from a genetic database.
(ii) Description of Related Art
Certain combinations of nucleotides, when present in a polynucleotide, are known to give rise to certain properties in the polypeptide translated therefrom. One example includes those nucleotides which encode polypeptide hormone precursors which undergo a post-translation amidation reaction. Another example, as set forth in U.S. Pat. No. 4,917,999, relates to certain nucleotides which are characteristic of polypeptides exhibiting &agr;-amylase enzymatic activity.
Amidated polypeptide hormones are synthesized in the form of a precursor which undergoes maturation. This maturation consists of an amidation reaction. The amidation reaction of the C-terminal end is a characteristic reaction of amidated polypeptide hormones. This reaction, which occurs on the precursor of one or more hormones, allows maturation of the hormone and also ensures its biostability in the physiological medium: the amide group formed is less vulnerable than the free acid function. The hormone is therefore more resistant to carboxypeptidases, it remains active in the cell for longer and retains an optimum affinity for its receptor site.
Amidation has been widely described (“Peptide amidation”, Alan F. Bradbury and DerekG. Smyth, TIBS 16:112-115, March 1991 and “Functional and structural characterization of peptidylamidoglycolate lyase, the enzyme catalysing the second step in peptide amidation”, A. G. Katopodis, D. S. Ping, C. E. Smith and S. W. May, Biochemistry, 30(25): 6189-6194, June 1991), and its mechanism is as follows:
1—Cleavage of the precursor polypeptide chain of the hormone by an endoprotease at the two basic amino acids, that is to say arginine and/or lysine,
2—Subsequently two cleavages by carboxypeptidase result, which lead to the extended glycine intermediate,
3—The enzyme PAM (peptidyl-glycine-amidating monooxygenase) comprises two distinct enzymatic activities: firstly, it converts the extended glycine intermediate into an &agr;-hydroxyglycine derivative, the subunit of the enzyme PAM involved is PHM (peptidyl-glycine-hydrolylating monooxygenase). The derivative obtained serves as the substrate for the second subunit of PAM (called PAL: peptidyl-hydroxyglycine-amidating lyase), which fixes the amine function of the glycine on to the amino acid immediately adjacent to the N-terminal side and liberates glyoxylate.
This reaction involves the presence of a recognition site on the precursor of the hormone or hormones, a site which always comprises the sequence: glycine and two basic amino acids (arginine or lysine). The amidated polypeptide hormones which are to be secreted outside the endoplasmic reticulum are known to comprise a consensus signal sequence of about fifteen to thirty amino acids, this sequence being present at the N-terminal end of the polypeptide chain. It is cut later by a signal peptidase enzyme such that it is no longer found in the protein once secreted.
Given the importance of known amidated polypeptides in the context of numerous biological systems, methods have been sought for the identification of additional amidated polypeptides. Unfortunately, at the present time, the discovery of a new protein is not easy.
To date, the art has developed certain approaches in an attempt to identify novel proteins of potential biological interest.
In one approach, potentially new proteins of interest are isolated from a source by selecting a specific property which the researcher believes will be possessed by one or more potential proteins of interest in a sample. According to this approach, proteins can be isolated and purified by various techniques: precipitation at the isoelectric point, selective extraction by certain solvents and then purification by crystallization, counter-current distribution, adsorption, partition or ion exchange chromatography, electrophoresis.
The conventional protein isolation techniques described above provide only limited success in the isolation and identification of new biological molecules of interest. This approach implies knowledge of the properties of the protein to be isolated. Typically, one of two situations arises based on isolation of proteins using a common property. In the first situation, the common property will be for the most part unrelated or only marginally related to the biological function of the molecules being isolated. One could envision, for example, two proteins sharing identical isoelectric points but having completely unrelated biological functions. In the second situation, separation might be achieved based on common property which is very closely related to the biological function of the molecule being isolated. In this category, for example, one might envision molecules which bind to the same receptor molecule. In the former situation, the isolation of potentially new polypeptides is quite unfocussed given its likelihood of isolating compounds of completely unrelated biological function. By complete contrast, the latter situation suffers the exact opposite deficiency in that it enables isolation of only a very limited number of new biologically interesting molecules.
Thus, a person skilled in the art seeking to isolate potentially new polypeptides of interest by conventional protein separation techniques was confronted with the dilemma of obtaining a hodgepodge of biologically unrelated polypeptides or, alternatively, only a very specific set of polypeptides.
Another serious shortcoming of conventional techniques for isolation of new polypeptides from a sample relates to the nature of the sample itself. Obviously, there will be a limited number of available polypeptides for isolation and identification in any given biological sample. Furthermore, great care must be taken with such samples to ensure the continued integrity of the biologically active molecules therein.
Not surprisingly, previous attempts to isolate and characterize new peptides comprising an amidated C-terminal end have followed the conventional approach of starting with a biological sample and choosing from the arsenal of known separation techniques for isolating and identifying the peptides. For example, in U.S. Pat. No. 5,360,727 in the name of Matsuo et al., there was isolated a C-terminal alpha-amidating enzyme of porcine origin by extracting and purifying the enzyme from porcine atrium cords exhibiting the enzyme activity. In U.S. Pat. No. 5,871,995 issued in the name of lida et al., purified enzymes participating in C-terminal amidation were purified from a biological material such as horse serum by affinity chromatography using a peptide C-terminal glycine adduct as a ligand. In U.S. Pat. No. 4,708,934 in the name of Gilligan et al., peptidyl-glycine alpha.-amidating monooxygenase enzyme was extracted from medullary thyroid carcinoma cell lines and tissue samples. Where identification of substantial numbers of new polypeptides capable of amidation is the goal, conventional isolation techniques such as these are completely unsuitable, as they typically permit isolation of only a single polypeptide of interest from a source suspected to contain that polypeptide.
In PCT/FR98/01767, the assignee of the present application has recently developed a method which overcomes many of disadvantages discussed above in that it enables the rapid identification of a large number of putative peptides which comprise an amidated C-terminal end. In particular, unlike earlier techniques which relied on a particular physical property of the polypeptide to isolate it from a source suspected or known to contain it, the method developed by the assignee relies on a characteristic of the peptide sequence of the precursor of all amidated hormones known to
Bergé Gilbert
Camara y Ferrer Jose Antonio
Gozé Catherine
Martinez Jean
Thurieau Christophe Alain
Allen Marianne P.
Hunton & Williams
Sheinberg Monika
Societe de Conseils de Recherches et d'Applications Scienti
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