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 - 13 . (canceled) 
     
     
         14 . A nucleic acid encoding 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. 
     
     
         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 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. 
     
     
         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 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. 
     
     
         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.