[특허]Catalytically active recombinant memapsin and methods of use thereof

Catalytically active recombinant memapsin and methods of use thereof 원문보기

IPC분류정보
국가/구분	United States(US) Patent 등록
국제특허분류(IPC7판)	C07K-001/00
출원번호	UP-0888920 (2007-08-03)
등록번호	US-7829669 (2010-11-25)
발명자 / 주소	Koelsch, Gerald Tang, Jordan J. N. Hong, Lin Ghosh, Arun K. Lin, Xinli
출원인 / 주소	Oklahoma Medical Research Foundation The Board of Trustees of the University of Illinois
대리인 / 주소	Morrison & Foerster LLP
인용정보	피인용 횟수 : 4 인용 특허 : 69

초록 ▼

Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1). The inhibition constant of OM99-2 is 1.6×10−9 M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bond to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer's disease.

대표청구항 ▼

What is claimed is: 1. A memapsin 2 protein consisting of SEQ ID NO: 40. 2. The memapsin 2 protein of claim 1, wherein the memapsin 2 protein is expressed in a bacterial cell. 3. The memapsin 2 protein of claim 1, wherein the memapsin 2 protein is refolded after expression in a bacterial cell. 4. The memapsin 2 protein of claim 3, wherein the memapsin 2 protein is refolded by dissolution in a urea solution having a urea concentration of about 8M and at least one reducing agent at a pH of at least about pH 8.0. 5. The memapsin 2 protein of claim 4, wherein the memapsin 2 protein is refolded by dilution in an aqueous buffer having a pH of at least about pH 9, and wherein the pH subsequently is adjusted to about pH 8, and then maintained at a temperature of about 4° C. for a period of time in a range of between about 24 hours and about 48 hours. 6. The memapsin 2 of claim 4, wherein the memapsin 2 protein is refolded by dilution in an aqueous buffer having a pH of at least about pH 9 and including an oxidized and reduced glutathione, and wherein the urea concentration of the diluted solution of memapsin 2 protein decreases to about 4 M, and wherein the pH is adjusted to about pH 8. 7. The memapsin 2 protein of claim 4, wherein the memapsin 2 protein is refolded by dissolution in urea having a urea concentration of about 8 M and a pH of about pH 10, wherein a resulting solution subsequently is diluted in an aqueous buffer having a pH of about pH 9.0, and maintaining the solution at a temperature of about 4° C. for a period of time in a range of between about 24 hours and about 48 hours.

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