RECOMBINANT NELL PROTEIN PRODUCTION
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.