Purification of HER-2 variants
The present invention provides for a novel method for purification of EGFR family proteins obtained from cultures of insect cells. The process comprises subsequent steps of a) diafiltration and exchange of culture medium with buffer, b) immobilized metal affinity chromatography (IMAC), c) size exclusion chromatography (SEC), and d) anion exchange chromatography (AIE). The method also provides for an immunogenic variant of HER-2 protein for which the purification process has been especially adapted, as well as means for the preparation of the variant.
1. A nucleic acid fragment that encodes a polypeptide that comprises the amino acid sequence set forth in SEQ ID NO: 2, residues 17-677.
2. The nucleic acid fragment according to claim 1 , which is a DNA fragment.
3. A vector carrying the nucleic acid fragment according to claim 2 .
4. The vector according to claim 3 , which is capable of autonomous replication.
5. The vector according to claim 4 being selected from the group consisting of a plasmid, a phage, a cosmid, and a virus.
6. The vector according to claim 3 , which is an expression vector.
7. The vector according to claim 6 , comprising in the 5′→3′ direction and in operable linkage, a promoter for driving expression of the nucleic acid fragment, a nucleic acid sequence encoding a leader peptide enabling secretion of or integration into the membrane of the encoded polypeptide, and the nucleic acid fragment.
8. A transformed host cell carrying the vector of claim 3 .
9. A transformed host cell carrying the vector of claim 6 .
10. A stable cell line which carries the vector according to claim 6 .
11. A composition comprising the vector according to claim 6 in admixture with a pharmaceutically acceptable carrier or vehicle and optionally an adjuvant.
12. A nucleic acid fragment that encodes a polypeptide that consists of the amino acid sequence set forth in SEQ ID NO: 2, residues 17-677.