INGAP protein involved in pancreatic islet neogenesis

Abstract

Cellophane wrapping (CW) of hamster pancreas induces proliferation of duct epithelial cells followed by endocrine cell differentiation and islet neogenesis. Using the mRNA differential display technique a cDNA clone expressed in cellophane wrapped but not in control pancreata was identified. Using this cDNA as a probe, a cDNA library was screened and a gene not previously described was identified and named INGAP.

1. A preparation of a naturally occurring mammalian islet neogenesis associated protein (INGAP protein) substantially free of other mammalian proteins, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 . 
     
     
       2. The preparation of  claim 1  wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 
     
     
       3. A preparation of a polypeptide which comprises a sequence of at least 15 consecutive amino acids of a naturally occurring mammalian islet neogenesis associated protein (INGAP protein), wherein said polypeptide has immunogenic activity and wherein said sequence is a portion of INGAP protein, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 . 
     
     
       4. The preparation of  claim 3  wherein said  A polypeptide which is a fusion of said  ( 1 )  a first  sequence of at least  15  consecutive amino acids of a naturally occurring mammalian islet neogenesis associated protein ( INGAP protein ) , wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 , wherein said first sequence has immunogenic activity  to ( 2 ) a second polypeptide  sequence derived from a second protein. 
     
     
       5. The preparation of  claim 3  wherein said polypeptide is conjugated to a second polypeptide. 
     
     
       6. The preparation of  claim 3  wherein said polypeptide is conjugated to a solid support. 
     
     
       7. The preparation of  claim 3  wherein said polypeptide has a biological activity of said mammalian INGAP protein  is capable of stimulating beta cell regeneration in pancreatic ductal cells. 
     
     
       8. The preparation of claim  7    3  wherein said biological activity is  the polypeptide has the ability to stimulate pancreatic duct cells to grow and proliferate. 
     
     
       9. The preparation of  claim 3  wherein said polypeptide comprises amino acids #103 to #122 of the mammalian INGAP protein as shown in SEQ ID NO:2. 
     
     
       10. The preparation of  claim 3  wherein said polypeptide comprises at least 130 consecutive amino acids of said mammalian INGAP protein as defined by SEQ ID NO:2. 
     
     
       11. A preparation of an islet eogenesis  neogenesis associated protein (INGAP protein) of a mammal according to  claim 1 , substantially purified from other proteins of the mammal  wherein said INGAP protein is inducible upon  made by the process of inducing production of said protein by cellophane-wrapping of pancreas of the mammal. 
     
     
       12. A pharmaceutical composition for treatment of pancreatic insufficiency, comprising:
 a naturally occurring mammalian islet neogenesis associated protein (INGAP protein) in a pharmaceutically acceptable diluent or carrier, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 .  
 
     
     
       13. The pharmaceutical composition of  claim 12  wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO: 2. 
     
     
       14. A pharmaceutical composition comprising:
 a preparation of a polypeptide which comprises a sequence of at least 15 consecutive amino acids of a naturally occurring mammalian islet neogenesis associated protein (INGAP protein) and a pharmaceutically acceptable diluent or carrier, wherein said polypeptide is capable of stimulating β cell regeneration of pancreatic ductal cells, wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 , and wherein said sequence is a portion of said INGAP protein.  
 
     
     
       15. The  A pharmaceutical composition of  claim 14  wherein said polypeptide is a fusion of said sequence to a second polypeptide derived from a second protein  comprising:
   a preparation of a fusion polypeptide which is a fusion of  (   1   )  first sequence of at least  15  consecutive amino acids of a naturally occurring mammalian islet neogenesis associated protein  ( INGAP protein ) , wherein the first sequence is capable of stimulating β cell regeneration of pancreatic ductal cells, to  (   2   )  a second sequence derived from a second protein; and    
   a pharmaceutically acceptable diluent or carrier,    
   wherein said fusion polypeptide is capable of stimulating β cell regeneration of pancreatic ductal cells, and wherein the INGAP protein has the amino acid sequence shown in SEQ ID NO:  2 .    
 
     
     
       16. The pharmaceutical composition of  claim 14  wherein said polypeptide is conjugated to a second polypeptide. 
     
     
       17. The pharmaceutical composition of  claim 14  wherein said polypeptide has a biological activity of said mammalian INGAP protein. 
     
     
       18. The pharmaceutical composition of  claim 17  wherein said biological activity is the ability to stimulate pancreatic duct cells to grow and proliferate. 
     
     
       19. The pharmaceutical composition of  claim 14  wherein said polypeptide comprises amino acids #103 to #122 of the mammalian INGAP protein as shown in SEQ ID NO:2. 
     
     
       20. The pharmaceutical composition of  claim 14  wherein said polypeptide comprises at least 130 consecutive amino acids of said mammalian INGAP protein as defined by SEQ ID NO:2. 
     
     
       21. The preparation of  claim 1  which is free of other mammalian proteins. 
     
     
       22. The preparation of  claim 11  which is free from other proteins of the mammal. 
     
     
       23. The preparation of  claim 11  wherein the INGAP protein has 174 amino acids. 
     
     
       24. The preparation of  claim 11  wherein the INGAP protein is purified utilizing antibodies which immunoreact with INGAP. 
     
     
       25. The preparation of  claim 1  wherein the INGAP protein is made by a process of expression in a host cell which comprises a vector which comprises a nucleotide sequence which encodes the INGAP protein. 
     
     
       26. The preparation of  claim 3  wherein said polypeptide is made by a process of expression in a host cell which comprises a vector which comprises a nucleotide sequence which encodes the polypeptide. 
     
     
       27. The pharmaceutical composition of  claim 12  wherein said INGAP protein is substantially free of other mammalian proteins. 
     
     
       28. The pharmaceutical composition of  claim 14  wherein said polypeptide is substantially free of other mammalian protein. 
     
     
       29. A preparation of a polypeptide which comprises a sequence of at least  15  consecutive amino acids shown in SEQ ID NO:  2 , wherein said sequence is a portion of islet neogenesis associated protein ( INGAP protein )  capable of stimulating β cell regeneration of pancreatic ductal cells.   
     
     
       30. The preparation of  claim 29  wherein the polypeptide comprises a sequence of at least  15  consecutive amino acids selected from amino acids # 103  to # 122  as shown in SEQ ID NO:  2 . 
     
     
       31. A pharmaceutical composition comprising a preparation of a polypeptide which comprises a sequence of at least  15  consecutive amino acids shown in SEQ ID NO:  2 , wherein said sequence is capable of stimulating β cell regeneration in pancreatic ductal cells, wherein said sequence is a portion of islet neogenesis associated protein ( INGAP protein ). 
     
     
       32. The pharmaceutical composition of  claim 31  wherein the polypeptide comprises a sequence of at least  15  consecutive amino acids selected from amino acids # 103  to # 122  as shown in SEQ ID NO:  2 . 
     
     
       33. A preparation of a polypeptide which consists of a portion of islet neogenesis associated protein ( INGAP protein )  of at least  15  consecutive amino acids shown in SEQ ID NO:  2 ; wherein said polypeptide is capable of stimulating β cell regeneration of pancreatic ductal cells.   
     
     
       34. The preparation of  claim 33  wherein the polypeptide consists of a portion of islet neogenesis associated protein ( INGAP protein )  of at least  15  consecutive amino acids selected from amino acids # 103  to # 122  as shown in SEQ ID NO:  2 .   
     
     
       35. A pharmaceutical composition comprising a polypeptide which consists of a portion of islet neogenesis associated protein ( INGAP protein )  of at least  15  consecutive amino acids shown in SEQ ID NO:  2 , wherein said polypeptide is capable of stimulating β cell regeneration in pancreatic ductal cells.   
     
     
       36. The pharmaceutical composition of  claim 35  wherein the polypeptide consists of a portion of islet neogenesis associated protein ( INGAP protein )  of at least  15  consecutive amino acids selected from amino acids # 103  to # 122  as shown in SEQ ID NO:  2 .