Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2000-12-26
2004-04-13
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000
Reexamination Certificate
active
06720161
ABSTRACT:
This application is a 371 of PCT/EP99/02688 filed Apr. 21, 1999 which claims priority to Germany 198 18 077.2 filed Apr. 22, 1998.
Electrophoresis using gels of polyacrylamide, agarose, starch, etc., is state of the art for high resolution separation of complex mixtures of proteins, e. g., of cell extracts or of synthetic reaction mixtures (chemical synthesis, biological synthesis such as in vitro translation); (cf. G. M. Rothe, Electrophoresis of Enzymes—Laboratory Models, 1994, Springer Verlag, Berlin). Electrophoretical separation methods comprise electrophoresis, isotachophoresis, and isoelectric focussing. In particular, electrophoresis is conducted in a denaturating medium (in the presence of SDS, urea, guanidinium salts, mercaptoethanol, etc.) whereby the protein chains unfold and a separation mainly according to the size of the molecules is achieved. This denaturation, of course, eliminates any biological activity (ligand binding, substrate binding and substrate transformation) of the protein components. However, proteins can be refolded to their active, natural structure by a renaturation procedure. This renaturation can be conducted directly in the gel (ibid, p. 135 ff.). If the proteins are large compared to the pore structure of the gel matrix then the proteins remain localized at the separation position without substantially reducing the resolution of separation. The enzyme proteins can then develop their activity with respect to a substrate which is diffused into the gel after electrophoretic separation and optional renaturation or which was added to the gel before its polymerization (ibid, p. 165, ff.). The product of this enzyme reaction can be used to locally characterize and identify the enzyme if this product also remains localized in the gel at the (same) position at which the reaction takes place. A large number of such reactions have been described (ibid, p. 141, ff.) for which, however, the following restrictions are valid:
a) The substrate is small compared to the pore structure of the gel and is diffused into the gel after electrophoretic separation and optional renaturation; the enzyme reaction is then detected by the formation of a colored, insoluble (i. e., locally precipitating) product.
b) the substrate is large compared to the pore structure of the gel and is added to the gel before its polymerization; the product of the enzyme reaction remains large and is detected accordingly or the substrate is degraded into small fragments which are eluted out of the gel and the enzyme activity is detected as the absence of the substrate at the respective position (negative staining).
The choice of employable substrate molecules for the method in these embodiments, however, is very restricted due to the various specific requirements the substrate and the product, respectively, have to fulfill. Many enzymes can convert small substrates as well and can convert a large variety of synthetically accessible substrates as well. The products of these reactions cannot be adapted to the above requirements for in situ detection in the gel. As a possibility to use such substrates in a flexible way, e. g., Kameshita and Fujisawa have described covalent binding with a large carrier molecule (Anal. Biochem., 1996, Vol. 237, pp. 198-203). This is based on the same principle as described in item b), namely physical entrapment of the substrate conjugate in the matrix of the gel.
To detect protein kinases short synthetic oligopeptides, which had a cysteine unit at the N-terminus, were covalently bound to various polyamino acids having a size of 23 000 to 198 200 Dalton using the bifunctional reagent N-(-maleimidocaproyl) succinimide. In this manner, it was possible to detect the catalytical subunits of the cAMP dependent protein kinase (PKA), CaM kinase II and CaM kinase IV together with the synthetic peptide substrates Kamptide (CLRRWSVA), C-Syntide-2 (CPLARTLSVAGLLPLKK) and CAMKAKS (CSQPSFQWRQPSLDVDVGD) in the gel after radioactive phosphorylation in the presence of [&ggr;-
32
P] ATP and autoradiography. (These peptides are given in the one-letter-code with the N-terminus on the left hand side and the C-terminus on the right hand side.) However, it was observed that the kind of carrier molecule, in this case: its composition of amino acid units, influences the use of the substrate and that the carrier molecule itself can function as a substrate, respectively. The second possibility is very undesirable under specific circumstances if the enzymes are to be detected selectively and specifically.
The object of the present invention is to provide an improved method and an improved gel for detecting and separating enzyme activities which overcomes the disadvantages of the state of the art and particularly overcomes the limitations connected with the choice of the substrate and the negative influence of the carrier molecules.
This object is achieved by a method for attaching substrate molecules to gel matrices characterized in that a substrate is covalently bound to a polymerizable monomeric or oligomeric constitutional unit of a gel and the resultant product, i.e., the constitutional units of the gel, is polymerized.
Preferably, the substrate is a natural or synthetic (oligo)peptide, a natural or synthetic (oligo)nucleotide or a DNA fragment.
The monomeric or oligomeric constitutional units of the gel comprise conventional monomers or polymerizable oligomers for the preparation of gels, such as acrylic acid monomers and/or acrylic acid oligomers and/or methacrylic acid monomers and/or methacrylic acid oligomers. However, as described above, all other conventional constitutional units can also be employed which can be polymerized to a gel.
Preferably for the polymerization, constitutional units of substrate and gel are mixed with constitutional units of gel which do not include substrate components. Preferably, the ratio of constitutional units of substrate and gel to constitutional units of unmodified gel is chosen so that the amount of substrate in the gel is sufficient to detect the enzyme reaction. The polymerization can be a conventional type of polymerization such as solution or bulk polymerization.
According to a particularly preferred embodiment of the method according to the present invention a spacer (a bifunctional spacing molecule) is incorporated between the substrate and the monomeric or oligomeric constitutional unit of the gel and is covalently bound to both components.
Such a construction has the following constitution:
MONOMER−(SPACER)
optional
−SUBSTRATE
MONOMER (M): e.g., acrylic acid, methacrylic acid, etc.
SPACER (Sp):—aminocarboxylic acid (e.g., C
2
to C
20
), oligonucleotide, oligopeptide, polyethylene glycol, etc.).
SUBSTRATE (Sub): synthetic peptide, oligonucleotide, small organic molecule, etc.
The spacer can be a spacer which is conventionally used in peptide and nucleotide chemistry, such as a natural or synthetic (oligo)peptide or a natural or synthetic (oligo)nucleotide.
According to the present invention, the product which optionally contains a spacer and which contains a constitutional unit of at least one substrate and least one gel unit is added to unmodified gel monomers or a solution of unmodified gel monomers, optionally mixed and then polymerized. During polymerization, the modified, monomeric or oligomeric unit containing at least one substrate and optionally at least one spacer is incorporated into the gel matrix just like the unmodified gel monomers and thereby the substrate is covalently bound to the gel matrix.
Furthermore, the present invention refers to a gel, which comprises immobilized substrates. This gel is preferably prepared as described above. It is particularly preferred that the gel is a separating gel.
The size of the pores and the cross-linking of the gel can be regulated in a conventional manner by varying the respective conditions of polymerization, such as the choice of the solvent, the concentration, the temperature, the use of a catalyst, etc.
An advantage of the localized attachment of the substra
Frank Ronald
Tegge Werner
Frommer & Lawrence & Haug LLP
Gesellschaft fuer Biotechnologische Forschung mbH
Gitomer Ralph
Santucci Ronald R.
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