Recombinant nel-like (nell) protein production

Abstract

The present invention provides a method and system for producing a NELL protein. The method and system comprise a CELL encoding a NELL protein or peptide and a non-insect secretory signal peptide.

1 .- 26 . (canceled) 
     
     
         27 . A method of purifying a NEL-like protein (NELL) peptide comprising:
 (a) providing a crude solution comprising the NELL peptide, and   (b) subjecting the crude solution to a chromatography process to obtain a purified NELL peptide, wherein the chromatography process uses a medium comprising a metal ion bearing two or more charges.   
     
     
         28 . The method of  claim 27 , wherein the chromatography process comprises contacting the crude solution to a chromatography membrane or column. 
     
     
         29 . The method of  claim 27 , wherein the method further comprises, prior to step (a):
 (i) transfecting a mammalian cell with a nucleic acid construct comprising a nucleic acid encoding the NELL peptide in frame with a nucleic acid encoding a heterologous, non-insect secretory signal peptide;   (ii) culturing the mammalian cell under conditions that permit expression of the NELL peptide; and   (iv) collecting media comprising the NELL peptide secreted from the cell to form the crude solution.   
     
     
         30 . The method of  claim 27 , further comprising testing bone formation-inducing activity of the purified NELL peptide. 
     
     
         31 . The method of  claim 27 , wherein the chromatography process comprises membrane or affinity chromatography. 
     
     
         32 . The method of  claim 27 , wherein the metal ion is calcium ion. 
     
     
         33 . The method of  claim 29 , wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide. 
     
     
         34 . The method of  claim 33 , wherein the animal secretory signal peptide is a mammalian secretory signal peptide. 
     
     
         35 . The method of  claim 34 , wherein the mammalian secretory signal peptide is a human secretory signal peptide. 
     
     
         36 . The method of  claim 29 , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. 
     
     
         37 . The method of  claim 29 , wherein the nucleic acid encoding the NELL peptide encodes NELL1 or NELL2. 
     
     
         38 . The method of  claim 37 , wherein the nucleic acid encoding NELL1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. 
     
     
         39 . The method of  claim 37 , wherein the nucleic acid encoding NELL2 comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.