Crystallization and Structure Determination of Glycosylated Human Beta Secretase, an Enzyme Implicated in Alzheimer's Disease

Abstract

An inhibitor bound form of human beta secretase, also known as memapsin 2 and BACE, particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system has been crystallized, and the three dimensional x-ray crystal structure has been solved to 3.2 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of human beta secretase activity.

1 . A method for crystallizing a human beta secretase molecule or molecular complex comprising:
 preparing purified human beta secretase in the presence of an inhibitor; and   crystallizing human beta secretase from a solution having a pH of about 3.5 to about 5.5.   
     
     
         2 . The method of  claim 1  wherein the salt is selected from the group of sodium chloride, ammonium sulfate, magnesium sulfate, lithium sulfate, and combinations thereof. 
     
     
         3 . The method of  claim 1  wherein the solution has a pH of about 4.0 to about 4.7. 
     
     
         4 . The method of  claim 1  wherein the solution comprises a buffer having a pK a  of about 3 to about 6. 
     
     
         5 . The method of  claim 1  wherein the glycol is selected from the group of PEG, PEG-MME, PEG-DME, polyoxyalkylenepolyamines, and combinations thereof. 
     
     
         6 . The method of  claim 1  wherein the solution further comprises a salt. 
     
     
         7 . The method of  claim 6  wherein the salt is present in a concentration of about 0.001 M to about 0.5 M. 
     
     
         8 . The method of  claim 1  wherein the solution includes up to about 40% by weight organic solvent. 
     
     
         9 . The method of  claim 8  wherein the organic solvent is DMSO. 
     
     
         10 . The method of  claim 1  wherein the solution further comprises up to about 40% by weight ethylene glycol or glycerol. 
     
     
         11 . The method of  claim 1  wherein the beta secretase is present at a concentration of about 1 mg/ml to about 80 mg/ml. 
     
     
         12 . The method of  claim 1  wherein the inhibitor is present at a concentration of about 0.1 to about 10 mM. 
     
     
         13 . The method of  claim 1  wherein the solution further comprises about 5% by weight to about 50% by weight of a glycol. 
     
     
         14 . The method of  claim 13  wherein the glycol is a monomeric or polymeric glycol. 
     
     
         15 . The method of  claim 1  wherein the human beta secretase is isolated from mammalian cells. 
     
     
         16 . The method of  claim 15  wherein the mammalian cells are CHO-K1 cells. 
     
     
         17 . The method of  claim 15  wherein the mammalian cells are HEK 293 cells. 
     
     
         18 . The method of  claim 1  wherein the human beta secretase is isolated from insect cells as part of the Baculovirus expression system. 
     
     
         19 . A crystal of beta secretase having the trigonal space group symmetry P3 2 21.