Nucleic acid molecules for optimized CAB-2 expression and their use
The invention provides for nucleic acid molecules encoding a CAB-2 protein and variants thereof. The invention also provides constructs, transformed eukaryotic cells, and methods of producing CAB-2 protein and variants thereof.
1 . A nucleic acid molecule consisting of a mutagenized sequence of SEQ ID NO: 1, wherein the mutation is selected from the group consisting of A (501)→G, A (504)→C or T, A (1170)→G, T (1173)→C, A, or G, T (1524)→C, A (1527)→G, T (1800)→C, and A (1803)→G, and combinations thereof.
2 . The nucleic acid molecule of claim 1 , wherein there are at least two mutations and wherein
(a) at least one mutation is selected from the group consisting of A (501)→G, A (504)→C or T, T (1524)→C, A (1527)→G, T (1800)→C, and A (1803)→G, and
(b) at least one mutation is selected from the group consisting of A (1170)→G, and T (1173)→C, A, or G.
3 . The nucleic acid molecule of claim 1 , wherein the nucleic acid molecule is selected from the group consisting of a nucleic acid with the sequence of SEQ ID NO: 2 and a nucleic acid with the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.
4 . A nucleic acid molecule encoding a protein of the amino acid sequence SEQ ID NO: 3, wherein expression of the nucleic acid molecule produces protein of SEQ ID NO: 3 at a level of at least 60% that of a nucleic acid molecule consisting of the sequence of SEQ ID NO: 2 or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ when transformed into CHO-K1 cells under identical conditions.
5 . A construct comprising control elements and the nucleic acid molecule of claim 1 operatively linked thereto for expression of the nucleic acid molecule to produce a protein of SEQ ID NO: 3.
6 . A construct comprising control elements and the nucleic acid molecule of claim 2 operatively linked thereto for expression of the nucleic acid molecule to produce a protein of SEQ ID NO: 3.
7 . A construct comprising control elements and the nucleic acid molecule of claim 3 operatively linked thereto for expression of the nucleic acid molecule to produce a protein of SEQ ID NO: 3.
8 . A eukaryotic cell transformed with a nucleic acid molecule of claim 1 .
9 . The eukaryotic cell of claim 8 , wherein the cell is a CHO-K1 cell.
10 . A eukaryotic cell transformed with a nucleic acid molecule of claim 2 .
11 . The eukaryotic cell of claim 10 , wherein the cell is a CHO-K1 cell.
12 . A eukaryotic cell transformed with a nucleic acid molecule of claim 3 .
13 . The eukaryotic cell of claim 12 , wherein the cell is a CHO-K1 cell.
14 . A eukaryotic cell transformed with a nucleic acid molecule of claim 4 .
15 . The eukaryotic cell of claim 14 , wherein the cell is a CHO-K1 cell.
16 . A method of producing CAB-2 protein comprising:
(a) providing cells transformed with a nucleic acid molecule of claim 1 , and
(b) culturing the transformed cells to produce CAB-2 protein.
17 . A method of producing CAB-2 protein comprising:
(a) providing cells transformed with a nucleic acid molecule of claim 2 , and
(b) culturing the transformed cells to produce CAB-2 protein.
18 . A method of producing CAB-2 protein comprising:
(a) providing cells transformed with a nucleic acid molecule of claim 3 , and
(b) culturing the transformed cells to produce CAB-2 protein.
19 . A method of producing CAB-2 protein comprising:
(a) providing cells transformed with a nucleic acid molecule of claim 4 , and
(b) culturing the transformed cells to produce CAB-2 protein.
20 . The nucleic acid molecule of claim 4 wherein the nucleic acid has at least one base mutation in a site selected from the group consisting of:
(a) bases 501 to 506 of SEQ ID NO:1;
(b) bases 1170 to 1173 of SEQ ID NO:1;
(c) bases 1522 to 1527 of SEQ ID NO:1;
(d) bases 1665 to 1670 of SEQ ID NO:1; and
(e) bases 1798 to 1803 of SEQ ID NO:1.