Method of identification of novel substrates

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...

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435 71, 435 76, 435 18, C12Q 100

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active

060430230

ABSTRACT:
The present invention provides a novel method for the rapid isolation and identification of large numbers of novel enzyme substrates. The novel method provided by the present invention identifies substrates in tissue and/or cell extracts of a non-human model characterized as having an inactive enzyme. Without active enzyme, the substrates of this enzyme accumulate in the non-human model. The tissue or cell extract containing the enzyme substrate is then fractionated by passing the extract through an affinity column. The affinity column comprises an enzyme having similar specificity to the inactive enzyme, bound to a solid support. The affinity resin binds the enzyme substrate so that the substrate may be isolated from other proteins in the extract. The substrate of the enzyme may then be further identified by purifying and sequencing. Also provided by the present invention are novel substrates, and analogs of the substrates identified by the method of the present invention. The novel substrates are then processed and used in the development of therapeutics, such as in gene therapy and vaccines. Also provided by the present invention are receptors that bind to the protein, polypeptide, and peptide substrates of the present invention, and variations of these substrates for use in the development of drugs.

REFERENCES:
patent: 5624818 (1997-04-01), Eisenman et al.
Hibi, M. et al., "Indentification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain," Genes and Development (Nov. 1993), vol. 7, No. 11, pp. 2135-2148.
McLaughlin, M.M. et al. "Indentification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSPB p38 MAP kinase" The Journal of Biological Chemistry (Apr. 5, 1996), vol. 271, No. 14, pp. 8488-8492.
Varlamov, O. et al. "Induced and spontaneous mutations at Ser202 of carboxypeptidase E" The Journal of Biological Chemistry (Jun. 14, 1996), vol. 271, No. 24, pp. 13981-13986.

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