Production of factor VIIa/TF and factor Xa binding proteins

Abstract

A method for the production of Tissue Factor Pathway Inhibitor (TFPI) and Tissue Factor Pathway Inhibitor 2 (TFPI-2), and muteins thereof is disclosed wherein the protein is retained within a yeast cell during growth of the yeast cell and recovered from an insoluble fraction prepared from yeast cells containing the protein.

1 - 11 . (canceled)  
     
     
         12 . A method for producing a protein capable of binding a factor VIIa/TF complex, comprising: 
 (a) transforming yeast cells with a vehicle comprising a first nucleotide sequence encoding a protein comprising the amino acid sequence of TFPI shown in SEQ ID NO:3 or a mutein thereof;    (b) incubating the transformed yeast cells under conditions favorable for production of the protein, wherein the protein is retained within the yeast cells;    (c) preparing an insoluble fraction of the transformed yeast cells containing the protein; and    (d) recovering the protein from the insoluble fraction.    
     
     
         13 . The method of  claim 12  wherein the amino acid sequence of the protein consists essentially of the amino acid sequence of TFPI shown in SEQ ID NO:3.  
     
     
         14 . The method of  claim 12  wherein the protein is a mutein of TFPI.  
     
     
         15 . The method of  claim 12  wherein the protein consists essentially of a mutein of TFPI.  
     
     
         16 . The method of  claim 12  wherein the first nucleotide sequence is preceded in frame by a second nucleotide sequence, wherein said first and said second nucleotide sequences together encode a fusion protein capable of being cleaved within the yeast cells to release the protein capable of binding the factor VIIa/TF complex.  
     
     
         17 . The method of  claim 16  wherein the second nucleotide sequence encodes ubiquitin.  
     
     
         18 . The method of  claim 16  wherein the second nucleotide sequence encodes superoxide dismutase.  
     
     
         19 . A method for producing a protein capable of binding factor Xa, comprising: 
 (a) transforming yeast cells with a vehicle comprising a first nucleotide sequence encoding a protein comprising the amino acid sequence of TFPI shown in SEQ ID NO:3 or a mutein thereof;    (b) incubating the transformed yeast cells under conditions favorable for production of the protein, wherein the protein is retained within the yeast cells;    (c) preparing an insoluble fraction of the transformed yeast cells containing the protein; and    (d) recovering the protein from the insoluble fraction.    
     
     
         20 . The method of  claim 19  wherein the amino acid sequence of the protein consists essentially of the amino acid sequence of TFPI shown in SEQ ID NO:3.  
     
     
         21 . The method of  claim 19  wherein the protein is a mutein of TFPI.  
     
     
         22 . The method of  claim 19  wherein the protein consists essentially of a mutein of TFPI.  
     
     
         23 . The method of  claim 19  wherein the first nucleotide sequence is preceded in frame by a second nucleotide sequence, wherein said first and said second nucleotide sequences together encode a fusion protein capable of being cleaved within the yeast cells to release the protein capable of binding the factor Xa.  
     
     
         24 . The method of  claim 23  wherein the second nucleotide sequence encodes ubiquitin.  
     
     
         25 . The method of  claim 23  wherein the second nucleotide sequence encodes superoxide dismutase.