Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1999-11-15
2003-04-08
Naff, David M. (Department: 1651)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C435S091500, C435S180000, C435S181000, C435S455000, C435S803000, C435S815000, C530S326000, C530S350000, C530S815000, C530S816000
Reexamination Certificate
active
06545132
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a microsphere which is prepared by coupling a substance possessing physiological activities to a styrene-glycidyl methacrylate polymer through a spacer as well as a process of isolating an objective or targeted substance by using the microsphere of the invention.
2. Background
Cells constituting a living body are exposed to various kinds of stimulation from the external environment all the time. To respond to such stimulation the cells lead some gene groups to expression. As a result, various living phenomena can occur, such as induction of cell growth and/or cell differentiation and maintenance of physiological homeostasis. Extracellular stimulation is transformed into an intracellular signal, which activates a specific proteinous transcription factor. The functionally activated transcription factor binds to a specific base sequence on a chromosome to induce a gene group under its regulation to expression. The product of the induced gene expression primarily functions to respond to the stimulation in some cases. In the other cases, the product of the induced gene expression further activates another transcription factor that induces another gene group under its regulation to expression to secondarily respond to the stimulation. In either case, cellular response to the stimulation from the external environment is concluded to be functional transformation of transcription factors.
In recent years, an extremely interesting fact was revealed. That is, mechanisms of action of cyclosporin A (CysA) and FK506, immunosuppressive drugs, have been revealed. See J. Lin et al.,
Cell,
66:807-815 (1991); S. J. O'Keefe et al.,
Nature,
357:692 (1992); and N. A. Clipstone et al.,
Nature,
357:695 (1992). The first opportunity for revealing the mechanisms is the identification of intracellular receptors to these drugs. See R. E. Handschumacher et al.,
Science,
226, 554; and J. J. Sekierka et al.,
J. Immunol.,
143:1580-1583 (1989). On the basis of these findings, a series of signaling pathway following stimulation by antigen was revealed in T-cell that is immunocompetent cell.
Accordingly, investigation and identification of intracellular receptors to drugs, as well as elucidation of signaling pathway, are expected to be further developed into developmental research of new drugs targeting the signaling pathway and research for novel drug designs.
Conventional methods of isolation and purification of intracellular receptors to drugs are fractionation of crude cell extracts by using various columns, followed by detection of factors binding to labeled drugs in each fraction. Therefore, two steps of procedure, the first one was isolation and purification using columns and the second one was assay for binding activity against drugs, have been necessarily performed until now.
Accordingly, for the purpose of purification, identification and functional analysis of receptors to a specific drug, located within cells or on cellular membrane, certain drug-immobilized microspheres have been designed and constituted.
SUMMARY OF THE INVENTION
According to the conventional methods of purification, it can take an exceedingly long time to purify drug-binding factors from crude cell extracts and moreover, a yield of factors is quite low due to repeated fractionation using various columns. Therefore, a huge amount of starting material is necessary for the determination of an amino acid sequence of drug-binding factors. It also can be most difficult to establish an assay method for binding activity of receptors against drugs because obtained drug receptors are usually not identified. Conventional methods of determination of binding of receptors to drugs are filter binding method and gel filtration method which utilize the fact that drug receptors (proteins) bind to filters and that sizes of drugs binding to receptors become larger than free drugs and receptors. However, some receptors that do not bind to filters or other receptors change their conformation after binding to filters and discharge drugs. Therefore, properties of drug receptors should be preliminarily investigated in the conventional methods. The present invention aims at solving the above mentioned problems to provide drug-immobilized particles and a process of purifying proteins.
According to the present invention, a microsphere comprising styrene-glycidyl methacrylate polymer is provided, and isolation, purification and identification of receptors to a specific compound possessing physiological activities are easily performed.
In addition, the present invention is concerned with microspheres prepared by coupling substances and proteins purified using the microspheres of the invention.
The present invention is further concerned with microspheres comprising a substance possessing physiological activity, a polymer and a spacer, wherein at least one functional group of any of the substance possessing physiological activity, the polymer or spacer is converted to another type of functional group. The present invention also relates to a process of preparing such microspheres.
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S. O'Keefe et al.,Nature, 357:692 (1992).
N. Clipstone et al.,Nature, 357:695 (1992).
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Handa Hiroshi
Kawaguchi Haruma
Corless Peter F.
Edwards & Angell LLP
O'Day Christine C.
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