Claims
- 1. Process for improving the renaturation of recombinant proteins, comprising the steps of:
- adding at least one oligonucleotide sequence encoding a helper sequence of 2 to 20 amino acids in length to a DNA sequence coding for a protein, wherein said oligonucleotide sequence is added to the part of the DNA sequence encoding the protein's N and/or C-terminus, and wherein the relative hydrophobicity of said helper sequence, calculated as the sum of the relative hydrophobicities for the individual amino acids, is a negative numerical value, and wherein the helper sequences have a value for the ratio of relative hydrophobicity to the number of amino acids which is -2.0 kcal/mol or less,
- expressing the DNA sequence including the oligonucleotide sequence encoding said helper sequence, and
- thereafter renaturing any expressed protein.
- 2. Process according to claim 1, wherein the helper sequences have a value for the ratio of relative hydrophobicity to the number of amino acids which is -2.5 kcal/mol or less.
- 3. Process according to claim 1, wherein the oligonucleotide sequence is added to the part of the DNA sequence coding for the N-terminus of the recombinant protein.
- 4. Process according to claim 1, wherein the oligonucleotide sequence is added to the part of the DNA sequence coding for the C-terminus of the recombinant protein.
- 5. Process according to claim 1, wherein the helper sequences contain at least two amino acids selected from the group consisting of glutamate, aspartate, lysine, arginine and proline.
- 6. Process according to claim 5, wherein the helper sequences contain at least two glutamate residues.
- 7. Process according to claim 5, wherein the helper sequences contain two successive amino acids having the same charge selected from the group consisting of glutamate, aspartate, lysine and arginine.
- 8. Process according to claim 7, wherein the helper sequences contain two successive glutamate residues.
- 9. Process according to claim 1, further comprising the addition of a DNA fragment encoding a cleavage site between the oligonucleotide sequence encoding the helper sequence and the DNA encoding the protein.
- 10. Process according to claim 9, wherein the cleavage site is a sequence recognized by a protease.
- 11. Process according to claim 10, wherein the cleavage site is an IgA protease cleavage site.
- 12. Process according to claim 11, wherein the cleavage site is a factor Xa cleavage site.
- 13. Process according to claim 1, wherein the helper sequence is selected from the group consisting of:
- Met-Glu (SEQ ID:1)
- Met-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 2)
- Met-Thr-Pro-Leu-His-His-Pro-Arg-Pro-Pro (SEQ ID NO: 3)
- Met-Thr-Pro-Leu-Lys-Lys-Pro-Arg-Pro-Pro (SEQ ID NO: 4)
- Met-Thr-Pro-Leu-Glu-Glu-Gly-Pro-Arg-Pro-Pro (SEQ ID NO: 5)
- Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu-Pro-Arg-Pro-Pro (SEQ ID NO: 6)
- Met-Thr-Pro-Leu-Glu-Glu-Gln-Pro-Pro (SEQ ID NO: 7)
- Met-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His-Pro-Arg-Pro-Pro (SEQ ID NO: 8)
- Met-Thr-Pro-Leu-Glu-Glu-Gly-Ile-Glu-Gly-Arg (SEQ ID NO: 9) and
- Met-Thr-Pro-Leu-Lys-Ala-Lys-Arg-Phe-Lys-His-Pro-Arg-Pro-Pro (SEQ ID NO: 10).
- 14. The process according to claim 1, wherein said protein is selected from the group consisting of granulocyte colony stimulating factor (G-CSF) and a granulocyte stimulating factor (G-CSF) mutein, wherein one or more amino acids selected from the group consisting of (i) the amino acids of the sequence His-Ser-Leu at positions 52-54 of the mature G-CSF with 174 amino acids, (ii) the amino acids of the sequence His-Ser-Leu at positions 55-57 of the mature G-CSF with 177 amino acids, (iii) the His residues at positions 43, 79, 156 or 170 of the mature G-CSF with 174 amino acids and (iv) the His residues at positions 46, 82, 159 or 173 of the mature G-CSF with 177 amino acids are substituted with a different amino acid or deleted in said mutein.
Priority Claims (1)
Number |
Date |
Country |
Kind |
41 05 480.6 |
Feb 1991 |
DEX |
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Parent Case Info
This application is a continuation division of application Ser. No. 07/837,779 filed Feb. 14, 1992, now abandoned.
US Referenced Citations (10)
Foreign Referenced Citations (1)
Number |
Date |
Country |
389034 |
Sep 1990 |
EPX |
Non-Patent Literature Citations (2)
Entry |
Mulks et al. "Relationship Between the Specificity of IgA Proteases and Serotypes in Haemophilus influenzae", Journal of Infectious Diseases, vol. 146, No. 2, Aug. 1982, pp. 265-274. |
Mulks et al. (1980) Jour. Exp. Med. vol. 152 pp. 1442-1447. |
Continuations (1)
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Number |
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Parent |
837779 |
Feb 1992 |
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