Recombinant elastase proteins and methods of manufacturing and use thereof

Abstract

The present invention relates to methods for the manufacture, purification, formulation, and use of biologically active recombinant elastase proteins. Described are recombinant methods for producing therapeutically useful elastase proteins, as are pharmaceutical compositions comprising said elastase proteins. Novel recombinant elastase proteins and protein preparations are also disclosed. Methods are described for treating and preventing diseases of biological conduits using pharmaceutical compositions containing the elastase proteins of the invention.

1 . A proelastase protein comprising (i) optionally, a signal sequence; (ii) an elastase activation peptide sequence comprising an elastase recognition sequence operably linked to (iii) an amino acid sequence of a type I mature elastase. 
     
     
         2 . The proelastase protein of  claim 1 , wherein said elastase recognition sequence comprises the amino acid sequence of SEQ ID NO:119. 
     
     
         3 . The proelastase protein of  claim 2 , wherein proline is at the P2 position of said elastase recognition sequence. 
     
     
         4 . The proelastase protein of  claim 1 , wherein said elastase recognition sequence comprises the amino acid sequence of SEQ ID NO:124. 
     
     
         5 . The proelastase protein of  claim 4 , wherein proline is at the P2 position of said elastase recognition sequence. 
     
     
         6 . The proelastase protein of  claim 1  which comprises a cleavage domain the propeptide portion of which comprises the amino acid sequence of SEQ ID NO:120. 
     
     
         7 . The proelastase protein of  claim 4 , wherein proline is at the P2 position of said cleavage domain. 
     
     
         8 . The proelastase protein of  claim 4 , wherein histidine is at the P5 position of said cleavage domain. 
     
     
         9 . The proelastase protein of  claim 1  which comprises a cleavage domain of SEQ ID NO:123 or SEQ ID NO:125. 
     
     
         10 . The proelastase protein of  claim 1 , wherein said activation peptide sequence comprises the amino acid sequence of SEQ ID NO:121. 
     
     
         11 . The proelastase protein of  claim 1 , wherein the amino acid sequence of said type I mature elastase has at least 90% sequence identity to the amino acid sequence from position 6 to the end of SEQ ID NO:84 or SEQ ID NO:1. 
     
     
         12 . The proelastase protein of  claim 1  which comprises a signal sequence. 
     
     
         13 . The proelastase protein of  claim 1  which is isolated. 
     
     
         14 . A nucleic acid encoding a proelastase protein of  claim 1 . 
     
     
         15 . A vector comprising the nucleic acid molecule of  claim 14 . 
     
     
         16 . A host cell genetically engineered to express the nucleic acid molecule of  claim 14 . 
     
     
         17 . A solution comprising the proelastase protein of any one of  claim 1 . 
     
     
         18 . The solution of  claim 17  which is a buffer solution. 
     
     
         19 . The solution of  claim 18 , wherein the buffer solution comprises phosphate. 
     
     
         20 . The solution of  claim 18 , wherein the buffer solution comprises Tris base. 
     
     
         21 . A cell culture supernatant comprising the proelastase protein of  claim 1 . 
     
     
         22 . A method of producing a proelastase protein, comprising culturing the host cell of  claim 16  under conditions in which the proelastase protein is produced. 
     
     
         23 . The method of  claim 22 , further comprising recovering the proelastase protein. 
     
     
         24 . A method of producing a mature elastase protein, comprising subjecting the solution of  claim 17  to conditions that produce mature elastase protein. 
     
     
         25 . The method of  claim 24 , wherein said conditions do not include contacting said proelastase protein with trypsin. 
     
     
         26 . The method of  claim 24 , wherein said conditions include contacting said proelastase protein with a catalytic amount of an elastase. 
     
     
         27 . The method of  claim 24 , wherein said conditions include subjecting the solution to a pH of 6 to 11. 
     
     
         28 . The method of  claim 24 , wherein said solution comprises cell culture components. 
     
     
         29 . A method of producing a maturing elastase protein, comprising:
 (a) culturing the host cell of  claim 16  under conditions in which the proelastase protein is produced;   (b) recovering the proelastase protein, and   (c) subjecting a solution comprising said proelastase protein to conditions that produce a mature elastase protein,   
       thereby producing a mature elastase protein. 
     
     
         30 . The method of  claim 29 , further comprising the step of lyophilizing the mature elastase protein. 
     
     
         31 . The method of  claim 29 , further comprising the step of purifying the proelastase protein. 
     
     
         32 . The method of  claim 29 , further comprising the step of purifying the mature elastase protein.