METHODS OF INCERASING PROTEIN EXPESSION LEVELS

• US 20050074840A1
• Filed: 09/13/2004
• Published: 04/07/2005
• Est. Priority Date: 11/30/2001
• Status: Active Grant

• Current Assignee: Laboratoires Serono SA
• Original Assignee: Applied Research Systems ARS Holding NV (Merck & Co., Inc.)
• Inventors: Jiang, Xuliang, Schweickhardt, Rene Lynn, Brondyk, William
• Granted Patent: US 7,258,997 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: Y10S 930/144 Tumor necrosis factor

Abstract

Abstract of the Disclosure

The present invention relates to methods of increasing protein expression levels whereby at least one amino acid in a protein amino acid sequence is substituted for the amino acid, proline. Preferably, the substitution occurs within 15 amino acids, more preferably within 10 amino acids and most preferably within 5 amino acids of a cysteine amino acid residue. The present invention not only includes methods for polypeptides with proline substitutions, but also polynucleotides with codon substitutions for which a codon for any amino acid, except proline, is substituted for a codon encoding for proline.

Claims

1. A recombinant mutant of a native polypeptide, wherein the native polypeptide has at least one amino acid residue that has angles that are favored for proline, and wherein said mutant has at least one of said amino acid residues substituted by proline.

2. The polypeptide of claim 1, wherein the mutant exhibits increased folding efficiency.

3. The polypeptide of claim 2, wherein the substituted amino acid of the native polypeptide has a ( angle of –

45 to –

90 degrees and a ( angle of –

60 to 0 or 120 to 180 degrees.

4. The polypeptide of claim 3, wherein the native polypeptide comprises a cysteine residue capable of forming a disulfide bond, and wherein said substituted amino acid is within 15 residues of said cysteine residue.

5. The polypeptide of claim 4, wherein the substituted amino acid is within 10 residues of said cysteine residue.

6. The polypeptide of claim 5, wherein the substituted amino acid is within 5 residues of said cysteine residue.

7. A nucleic acid encoding the polypeptide of any one of claims 2-6.

12. An expression vector comprising the nucleic acid of claim 7, wherein said nucleic acid is operatively linked to an expression control sequence.

14. A method of producing an isolated mutant of a native polypeptide comprising:

(a) providing a host cell comprising the expression vector of claim 12; and

(b) expressing the mutant polypeptide.

8. A recombinant mutant of a native polypeptide, wherein the native polypeptide comprises the amino acid sequence of SEQ ID NO:

1, and wherein amino acid 34 or 87 of SEQ ID NO;

1 is substituted with proline.

9. The polypeptide of claim 8, wherein the amino acid at position 34 of SEQ ID NO:

1 is substituted with proline.

10. The polypeptide of claim 8, wherein the amino acid at position 87 of SEQ ID NO:

1 is substituted with proline.

11. A nucleic acid encoding the polypeptide of any one of claims 8-10.

13. An expression vector comprising the nucleic acid of claim 11, wherein said nucleic acid is operatively linked to an expression control sequence.

15. A method of producing an isolated mutant of a native polypeptide comprising:

(a) providing a host cell comprising the expression vector of claim 13; and

(b) expressing the mutant polypeptide.