Novel epidermal growth factor protein and gene, and methods of use therefor
AssigneeWaratah Pharmaceuticals, Inc.
InventorsSheila G. Magil, Susan D. Jones, Gary W. Pace, Stephen J. Brand
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Abstract
Forms of epidermal growth factor that are resistant to proteolysis, and gene sequences encoding these forms and having codons optimized for usage by an industrial production organism, are provided.
Claims84
A composition comprising an amino acid sequence of length X, X being an integer that is at least 48 and not more than 53, such sequence (i) being substantively homologous to a portion of SEQ ID NO:
- 1 from position 1 to position X-1 of SEQ ID NO:
- 1, and (ii) having at position X an amino acid residue different from that found in SEQ ID NO:
- 1.
A composition comprising an amino acid sequence substantially homologous to SEQ ID NO:
- 1, wherein the amino acid residue at position 51 of SEQ ID NO:
- 1 is an amino acid other than glutamic acid.
A composition comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:
- 2, 3 and 4.
A composition as shown in SEQ ID NO:
- 2.
A polypeptide of 51 amino acids in length, wherein residues 1-50 are substantially homologous to the amino acid sequence as shown in SEQ ID NO:
- 1, and residue 51 is an asparagine residue.
A composition comprising a human epidermal growth factor (EGF) having an amino acid sequence substantially homologous to that of at least positions 1-47 as shown in SEQ ID NO:
- 1, and having at least one amino acid replacement at positions 48-53 of the EGF carboxy terminus, the amino acid sequence being more stable to proteolysis than that of SEQ ID NO:
- 1.
A nucleotide sequence encoding a composition according to any of 1, 16, and 29, the nucleotide sequence having codons adjusted for optimum usage in an industrially acceptable production organism.
A polynucleotide having a nucleotide sequence encoding a polypeptide of 51 residues in length and having a biological activity of human EGF, the sequence containing codons that are optimized for expression in a species of Pichia, and having an amino acid at the carboxyl terminus capable of conferring resistance to proteolysis.
A recombinant strain of Pichia carrying a nucleotide sequence as shown in SEQ ID NO:
- 5.
A recombinant strain of Pichia capable of producing the amino acid sequence as shown in SEQ ID NO:
- 2.
A nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:
- 2.
A method of obtaining a composition comprising an amino acid sequence of length X, X being an integer that is at least 48 and not more than 53, such sequence (i) being substantively homologous to the portion of SEQ ID NO:
- 1 from position 1 to position X-1 of SEQ ID NO:
- 1, and (ii) having at position X an amino acid residue different from that found in SEQ ID NO:
- 1, the method comprising:
- designing a gene encoding the composition, having codons selected for optimum usage in an industrially acceptable organism;
- and
- producing the composition in the organism by fermentation of the organism.
A method of obtaining a modified amino acid sequence of an EGF that is more resistant to proteolysis than a nature identical EGF and that substantially retains a biological activity, the method comprising:
- identifying at least one residue of a sequence of the nature identical EGF that is subject to proteolysis by an industrially useful organism;
- designing the modified amino acid sequence in which the residue identified as subject to proteolysis is deleted or substituted with a different amino acid;
- and
- providing the modified amino acid sequence and testing proteolysis in comparison to the nature identical EGF, to obtain a modified EGF that is more resistant to proteolysis than the nature identical EGF.
Dependencies are inferred from the claim text.