Production of glucagon like peptide 2 and analogs

Abstract

GLP-2 peptides and analogs thereof are produced in high yield and with desired, authentic termini by isolation from a GLP-2 peptide multimer in which at least two units of GLP-2 peptide are coupled through a linker that presents an N-terminal acid cleavage site and a C-terminal enzyme cleavage site. In a specific embodiment, [Gly 2 ]hGLP-2 is produced from a multimeric precursor comprising 2-30 units thereof.

1 . A single chain protein multimer comprising at least two units of a GLP-2 peptide coupled tandemly by a linker that provides an acid cleavage site at the N-terminus of each GLP-2 unit, and an enzyme cleavage site at the C-terminus of each GLP-2 unit, wherein cleavage of said multimer with an acid and an enzyme liberates said GLP-2 peptide units, each having authentic N- and C-terminal residues. 
     
     
         2 . A multimeric protein according to  claim 1 , wherein said linker has the sequence ProValSerGlyProArg. 
     
     
         3 . A multimeric protein according to  claim 1 , wherein said GLP-2 peptide is [Gly 2 ]hGLP-2. 
     
     
         4 . A multimeric protein according to  claim 2 , wherein said GLP-2 peptide is [Gly 2 ]hGLP-2. 
     
     
         5 . A multimeric protein according to  claim 1 , further comprising a carrier protein coupled releasably at the N-terminus thereof, the carrier protein providing a Met residue at the N-terminus thereof and, at the C-terminus thereof, a site cleavable by said enzyme. 
     
     
         6 . A process for preparing a multimer according to  claim 1 , comprising the step of culturing a cellular host that incorporates an expression construct in which a DNA molecule coding for said multimer is linked operably with DNA providing for the expression thereof. 
     
     
         7 . The process according to  claim 6 , wherein said host is an  E. coli  host. 
     
     
         8 . The process according to  claim 6 , wherein the expression construct further comprises expression controlling elements of the T7 gene. 
     
     
         9 . DNA coding for multimer according to any one of  claim 1 . 
     
     
         10 . A process for preparing a GLP-2 peptide, comprising:
 (a) the steps of obtaining a multimer according to  claim 1 ,   (b) treating the multimer with acid and with enzyme to cleave linker resident therein, and   (c) isolating the resulting GLP-2 peptide units.   
     
     
         11 . The process according to  claim 10 , comprising:
 (a) first cleaving the multimer with acid,   (b) isolating the resulting cleaved multimer;   (c) cleaving with enzyme, and   (d) isolating the resulting GLP-2 peptide units having authentic termini.   
     
     
         12 . The process according to  claim 10 , wherein the acid cleavage step is performed at the time of extracting the GLP-2 peptide multimer from a cellular host producing said multimer. 
     
     
         13 . The method according to  claim 10 , wherein the step of treating the multimer with acid and with enzyme is performed without separation of reaction products prior to enzyme treatment. 
     
     
         14 . Recombinant [Gly2]hGLP-2, having a mass essentially identical to theoretical mass. 
     
     
         15 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically useful amount of recombinant [Gly 2 ]hGLP-2 having a mass essentially identical to theoretical mass.