Method for production of recombinant human thrombin

Abstract

The present invention relates to a method is provided for producing recombinant human thrombin from recombinant prothrombin using recombinant ecarin having the sequence SEQ ID NO 2 or a homologue thereof.

1 . A method for producing recombinant human thrombin from recombinant prothrombin using recombinant ecarin having the sequence SEQ ID NO 2 or a homologue thereof. 
     
     
         2 . A method according to  claim 1 , wherein the recombinant ecarin is being expressed and secreted by a cell containing the gene comprising the nucleotide sequence SEQ ID NO 2 or a homologue thereof in CHO-S cells, which ecarin has an amino acid sequence equal to that of wild type ecarin. 
     
     
         3 . A method according to  claim 1  or  2 , wherein recombinant protrombin is subjected to recombinant ecarin, which recombinant ecarin was isolated in active form after extra-cellular expression by CHO-S cells, said cells being left to apoptosis/necrosis for a time sufficient to activate said ecarin, whereupon a human recombinant thrombin is isolated. 
     
     
         4 . A method according to any of the preceding claims, wherein the recombinant prothrombin is produced by a cell-line comprising a prothrombin expressing gene having a nucleotide sequence comprising the sequence SEQ. ID. NO. 1. 
     
     
         5 . A method according to any of the preceding claims, wherein the recombinant prothrombin is a mixture of fully carboxylated prothrombin and incompletely carboxylated prothrombin. 
     
     
         6 . A method according to any of the preceding claims, wherein the recombinant prothrombin is a fully carboxylated prothrombin. 
     
     
         7 . A method according to any of the preceding claims, wherein the recombinant prothrombin is an incompletely carboxylated prothrombin. 
     
     
         8 . A recombinant thrombin obtained by the method according to any of the preceding claims. 
     
     
         9 . A pharmaceutical composition comprising a recombinant thrombin according to  claim 8 , in combination with pharmaceutically acceptable carriers, vehicles and/or adjuvants. 
     
     
         10 . A pharmaceutical composition according to  claim 9 , wherein the composition is in an applicable form. 
     
     
         11 . An isolated DNA sequence encoding ecarin according to SEQ ID NO 2 or a homologue thereof, having at least 80% identity to SEQ ID NO 2. 
     
     
         12 . An isolated DNA according  claim 11 , wherein the homologue has an identity of at least 90% to SEQ ID NO 2. 
     
     
         13 . A vector comprising the isolated DNA sequence of any of the  claims 11  to  12 . 
     
     
         14 . An amino acid sequence encoded by SEQ ID NO 2 or a homologue thereof. 
     
     
         15 . A host cell comprising the vector of  claim 13 . 
     
     
         16 . A host cell according to  claim 15 , wherein the host cell is a mammalian cell.