Recombinant 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 . A method of expressing a peptide in a mammalian cell comprising:
 providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a non-insect signal peptide; transfecting a mammalian cell with the nucleic acid construct; and culturing the mammalian cell under conditions that permit expression of the NELL peptide, wherein the non-insect signal peptide is a non-insect secretory signal peptide.   
     
     
         2 . The method of  claim 1 , wherein the mammalian cell is a Chinese hamster ovary (CHO) cell. 
     
     
         3 . The method of  claim 2 , wherein the nucleic acid encodes NELL1 or NELL2, wherein NELL1 is selected from the group comprising: 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, wherein NELL2 is 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. 
     
     
         4 . The method of  claim 2 , further comprising collecting NELL peptide secreted from the cell line; and
 substantially purifying the NELL peptide.   
     
     
         5 . The method of  claim 3 , further comprising collecting NELL peptide secreted from the cell line; and substantially purifying the NELL peptide. 
     
     
         6 . The method of  claim 5 , further comprising testing the activity of the NELL peptide to induce bone formation. 
     
     
         7 . The method of  claim 6 , further comprising testing the activity of the NELL peptide to induce bone formation. 
     
     
         8 . The method of  claim 1 , wherein the purifying includes chromatography purification. 
     
     
         9 . The method of  claim 1 , wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide, wherein the animal secretory signal peptide is a mammalian secretory signal peptide. 
     
     
         10 . A nucleic acid construct for expressing a NELL peptide in a mammalian cell, the nucleic acid construct comprising at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a non-insect signal peptide that is a non-insect secretory signal peptide. 
     
     
         11 . The nucleic acid construct of  claim 10 , wherein the nucleic acid encodes NELL1 or NELL2,
 wherein the NELL1 is selected from the group comprising: 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,   wherein NELL2 is 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.   
     
     
         12 . The nucleic acid construct of  claim 10 , wherein the mammalian cell is a CHO cell. 
     
     
         13 . The nucleic acid construct of  claim 10 , wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide, wherein the animal secretory signal peptide is a mammalian secretory signal peptide. 
     
     
         14 . A mammalian cell line for expressing a NELL peptide, the cell line including a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a non-insect signal peptide that is a non-insect secretory signal peptide. 
     
     
         15 . The cell of  claim 14 , wherein the mammalian cell line comprises a CHO cell. 
     
     
         16 . The cell of  claim 14 , wherein the CHO cell secretes NELL1 or NELL2 peptide. 
     
     
         17 . The cell of  claim 14 , wherein the nucleic acid encodes NELL or NELL2 peptide,
 wherein NELL1 is selected from the group comprising: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, and   wherein NELL2 is 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.   
     
     
         18 . The cell line of  claim 14 , wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide, wherein the animal secretory signal peptide is a mammalian secretory signal peptide. 
     
     
         19 . A recombinant peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and combinations thereof, and
 a non-insect secretory signal peptide.   
     
     
         20 . The combinant peptide of  claim 19 , wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide, wherein the animal secretory signal peptide is a mammalian secretory signal peptide. 
     
     
         21 . A method of purifying a NELL peptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to membrane chromotography to obtain a purified NELL peptide. 
     
     
         22 . The method of  claim 21 , wherein the membrane chromatography is anion-exchange membrane chromatography or cation-exchange membrane chromatography. 
     
     
         23 . The method of  claim 21 , wherein the NELL peptide is NELL-1 peptide or NELL-2 peptide. 
     
     
         24 . A method of purifying a NELLpeptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to chromotography process to obtain a purified NELL peptide, wherein the chromatography process comprises a medium comprising a metal ion bearing two or more charges. 
     
     
         25 . The method of  claim 24 , wherein the metal ion is calcium ion. 
     
     
         26 . The method of  claim 24 , wherein the NELL peptide is NELL-1 or NELL-2 peptide.