Method for autoactivation of procaspase 8

Abstract

A novel recombinant procaspase 8 protein lacking the death effector domains and a method for converting said novel procaspase protein to full active human caspase 8 by concentration of the procaspase 8 on a collection device.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding a human procaspase 8 polypeptide lacking the DED and having the complete amino acid sequence of SEQ ID NO:1; and    (b) a nucleotide sequence that is complementary to the nucleotide sequence of (a).    
     
     
         2 . The nucleic acid molecule of  claim 1 , wherein the polynucleotide molecule of 1 (a) comprises the complete nucleotide sequence of SEQ ID NO:2.  
     
     
         3 . An isolated nucleic acid molecule comprising a polynucleotide having a sequence at least 95% identical to a polynucleotide having the nucleotide sequence in (a) or (b) of  claim 1 .  
     
     
         4 . A vector comprising the nucleic acid molecule of  claim 1 .  
     
     
         5 . The vector of  claim 4 , wherein said nucleic acid molecule of  claim 1  is operably linked to a promoter for the expression of a human procaspase 8 polypeptide lacking the DED.  
     
     
         6 . A host cell comprising the vector of  claim 5 .  
     
     
         7 . The host cell of  claim 6 , wherein said host is  E. coli.    
     
     
         8 . A method of obtaining a human procaspase 8 polypeptide lacking the DED comprising culturing the host cell of  claim 7  and isolating said polypeptide.  
     
     
         9 . A human procaspase 8 polypeptide lacking the DED produced by the method of  claim 8 .  
     
     
         10 . An isolated human procaspase 8 polypeptide lacking the DED comprising the amino acid sequence of SEQ ID NO:1.  
     
     
         11 . A method for converting recombinant procaspase 8 protein lacking the DED to active human caspase 8 enzyme which comprises passing a solution of said protein through a matrix capable of concentrating said protein to affect auto-activation thereof to give active human caspase 8 enzyme.  
     
     
         12 . The method of  claim 11  wherein the procaspase 8 protein lacking the DED comprises the amino acid sequence of SEQ ID NO:1.  
     
     
         13 . The method of  claim 11  wherein the matrix is an ultrafiltration membrane.  
     
     
         14 . The method of  claim 11  wherein the matrix is a Ni chelate column.  
     
     
         15 . The method of  claim 11  wherein the matrix is an anion exchange column.  
     
     
         16 . The method of  claim 13  wherein the membrane is comprised of cellulose.  
     
     
         17 . The method of  claim 16  wherein the procaspase 8 protein lacking the DED comprises the amino acid sequence of SEQ ID NO:1.