Proteolytic Activation Of Recombinant Pro-memapsin 2
- Proteolytic Activation Of Recombinant Pro-memapsin 2017
- Proteolytic Activation Of Recombinant Pro-memapsin 2 0
[Show full abstract] activation mechanism and to produce stable mature memapsin 2 for kinetic/specificity studies, we have investigated the activation of recombinant pro-memapsin 2 by several.
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 analogs including isosteres at the sites of the critical amino acid residues were developed and the substrate analogs, OMR99-1 and OM99-2, were synthesized.
Proteolytic Activation Of Recombinant Pro-memapsin 2017
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. The inhibition constant of OM99-2 is 1.6×10 −9 M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bound 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. Purified recombinant catalytically active memapsin 2.
Proteolytic Activation Of Recombinant Pro-memapsin 2 0
The memapsin 2 of claim 1 having the amino acid sequence of SEQ ID NO.2 or the sequence present in a homologous species. The memapsin 2 of cliam 2 of human origin and having the amino acid sequence of SEQ ID NO.2. The memapsin 2 of claim 1 not including the transmembrane domain. The memapsin 2 of claim 1 expressed in a bacteria. The memapsin 2 of claim 1 cleaving SEVKM/DAEFR (SEQ ID NO:4) and SEVNL/DAEFR (SEQ ID NO:5) at pH 4.0 with k cat/K m of less than or equal to 39.9 s −1M −1 and less than or equal to k cat, 2.45 s −1, K m, 1 mM; k cat/K m, 2450 s −1M −1, respectively. A method for producing catalytically active recombinant memapsin 2 comprising refolding the recombinant memapsin 2 under conditions which dissociate and then slowly refold the enzyme into a catalytically active form.
The method of claim 7 wherein the memapsin 2 is first dissolved in 8 M urea solution including one or more reducing agents at a pH of greater than 8.0. The method of claim 8 wherein the memapsin 2 is then diluted into an aqueous buffer like 20 mM-Tris, pH 9.0, the pH slowly adjusted to approximately 8 with 1 M HCl, and the solution maintained at low temperature for approximately 24 to 48 hours before proceeding with purification. The method of claim 8 wherein the memapsin 2 is then rapidly mixed with an aqueous buffer like 20 mM-Tris, pH 9.0, containing oxidized and reduced glutathione, the process repeated, then the urea concentration decreased to approximately 0.4 M and the pH of the solution slowly adjusted to 8.0. The method of claim 8 wherein the memapsin 2 is dissolved in 8 M urea, pH 10.0, then rapidly diluted into an aqueous buffer like 20 mM Tris base, pH 9.0, and maintained at low temperature several hours, maintained at room temperature for several hours, and then the process repeated at decreasing pH. Dell driver download center.