Protocol for optimizing purification of enzymatically active human telomerase

Abstract

This invention provides purified telomerase and methods of purifying it. The methods involve the use of several sequential steps, including the use of matrices that bind molecules bearing negative charges, matrices that bind molecules bearing positive charges, intermediate-selectivity matrices, methods that separate molecules based on their size, shape, or buoyant density, and by affinity purification.

1 . A protocol for optimizing purification of human telomerase, comprising: 
 a) obtaining a solution of human telomerase;    b) measuring telomerase enzyme activity in the human telomerase solution;    c) combining the solution with a test affinity agent;    d) recovering mammalian telomerase that has bound to the test affinity agent;    e) measuring telomerase enzyme activity recovered from the affinity agent;    f) determining enrichment of the telomerase enzyme activity (in terms of telomerase activity per weight of protein) that results from binding and recovery of the human telomerase from the affinity agent; and    g) selecting the affinity agent tested in c) for use in purifying human telomerase in preference to other agents that are less effective for enriching the telomerase enzyme activity by the same criteria.    
     
     
         2 . The protocol of  claim 1 , wherein the test affinity agent is an oligonucleotide.  
     
     
         3 . The protocol of  claim 2 , wherein the oligonucleotide hybridizes with telomerase RNA component.  
     
     
         4 . The protocol of  claim 2 , wherein the oligonucleotide comprises a retrievable label such as biotin.  
     
     
         5 . The protocol of  claim 1 , wherein the solution of human telomerase obtained in step a) has been generated by a process comprising chromatography using a plurality of different matrices selected from anion exchange matrices, cation exchange matrices, and intermediate selectivity matrices.  
     
     
         6 . The protocol of  claim 1 , wherein the telomerase enzyme activity is determined by a primer elongation assay.  
     
     
         7 . The protocol of  claim 1 , wherein the telomerase enzyme activity is determined by a dot blot assay.  
     
     
         8 . The protocol of  claim 1 , whereby an affinity agent is identified in h) that improves purity of human telomerase by at least about 90-fold.  
     
     
         9 . A protocol for optimizing purification of human telomerase, comprising: 
 a) obtaining a solution of human telomerase;    b) measuring telomerase enzyme activity in the human telomerase solution;    c) enriching human telomerase in the solution by chromatographic separation using a test separation matrix;    d) combining the solution with an affinity agent;    e) recovering mammalian telomerase that has bound to the affinity agent;    f) measuring telomerase enzyme activity recovered from the affinity agent;    g) determining enrichment of the telomerase enzyme activity (in terms of telomerase activity per weight of protein) as a result of steps c), d), and e); and    h) identifying the separation matrix used in c) as improving effectiveness of said affinity agent in the purification of human telomerase if said matrix improves enrichment of telomerase enzyme activity recovered from the affinity agent.    
     
     
         10 . The protocol of  claim 9 , wherein the test matrix is a cation exchange matrix (such as a heparin matrix).  
     
     
         11 . The protocol of  claim 9 , wherein the test matrix is an anion exchange matrix.  
     
     
         12 . The protocol of  claim 9 , wherein the test matrix is an intermediate selectivity matrix bearing at least one of the following substituents: hydroxyapatite, a polyamine (such as spermine or spermidine), poly guanylic acid, a divalent metal ion (such as Ni ++ ), a positively charged poly-amino acid (such as poly-L lysine), a positively charged protein (such as histone), or aminophenyl-boronic acid.  
     
     
         13 . The protocol of  claim 9 , wherein the affinity agent is an oligonucleotide that hybridizes with telomerase RNA component.  
     
     
         14 . The protocol of  claim 9 , wherein the test separation matrix identified in h) improves the telomerase enzyme activity by at least 5.8-fold.  
     
     
         15 . A protocol for optimizing purification of human telomerase, comprising: 
 a) obtaining a solution of human telomerase;    b) measuring telomerase enzyme activity in the human telomerase solution;    c) enriching human telomerase in the solution by a test combination of sequential chromatographic separations using different matrices;    d) combining the solution with an affinity agent;    e) recovering mammalian telomerase that has bound to the affinity agent;    f) measuring telomerase enzyme activity recovered from the affinity agent;    g) determining enrichment of the telomerase enzyme activity (in terms of telomerase activity per weight of protein) as a result of steps c), d), and e); and    h) identifying the combination of chromatographic separation matrices tested in c) as improving effectiveness of said affinity agent in the purification of human telomerase if said combination improves enrichment of telomerase enzyme activity recovered from the affinity agent.    
     
     
         16 . The protocol of  claim 15 , wherein the combination of chromatographic separations identified in h) is performed using a plurality of different matrices selected from anion exchange matrices, cation exchange matrices, and intermediate selectivity matrices.  
     
     
         17 . The protocol of  claim 15 , wherein the combination of chromatographic separations identified in h) comprises separation using at least three different matrices.  
     
     
         18 . The protocol of  claim 15 , wherein the combination of chromatographic separations identified in h) improves the telomerase enzyme activity by at least 39-fold.  
     
     
         19 . A protocol for optimizing purification of human telomerase, comprising: 
 a) identifying a suitable affinity agent that specifically binds human telomerase by way of the method of  claim 1;  and    b) identifying a combination of chromatographic matrices that improves telomerase purification using said affinity agent by way of the method of  claim 15 .    
     
     
         20 . The protocol of  claim 19 , whereby a purification method is developed that produces human telomerase that is at least ˜2,000-fold more pure than a crude extract of 293 cells.