Novel epidermal growth factor protein and gene, and methods of use therefor

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.

1 - 43 . (canceled)  
     
     
         44 . A nucleotide sequence encoding a modified EGF polypeptide 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 at least 75% identical to that of positions 1-47 of SEQ ID NO: 1, and (ii) having at position X an amino acid residue different from that found in SEQ ID NO: 1, wherein said polypeptide comprises a biological activity that is at least 50% of that of the polypeptide whose sequence is shown in SEQ ID NO: 1, the activity being selected from the group consisting of mitogenesis, cell growth stimulation, tissue growth stimulation, cytoprotection, inhibition of acid secretion, migration stimulation, wound healing, growth of a tissue precursor cell, differentiation of a tissue precursor cell, growth and differentiation of a tissue precursor cell, and EGF receptor binding.  
     
     
         45 . A nucleotide sequence according to  claim 44 , wherein said nucleotide sequence comprises codons adjusted for optimum usage in yeast.  
     
     
         46 . A nucleotide sequence according to  claim 45 , wherein the yeast is  Pichia pastoris.    
     
     
         47 . 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.  
     
     
         48 . A recombinant strain of  Pichia  carrying a nucleotide sequence as shown in SEQ ID NO: 5.  
     
     
         49 . A recombinant strain of  Pichia  capable of producing the amino acid sequence as shown in SEQ ID NO: 2.  
     
     
         50 . A nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO: 2.  
     
     
         51 - 84 . (canceled)  
     
     
         85 . A nucleotide sequence according to  claim 44 , wherein the amino acid residue at position X is a neutral amino acid.  
     
     
         86 . A nucleotide sequence according to  claim 44 , wherein the amino acid residue at position X is aspargine, glutamine, alanine, or serine.  
     
     
         87 . A nucleotide sequence according to  claim 86 , wherein X is position 51.  
     
     
         88 . A nucleotide sequence according to  claim 86 , wherein said polypeptide has increased resistance to proteolysis in comparison with that of the polypeptide whose sequence is shown in SEQ ID NO: 1.  
     
     
         89 . A nucleotide sequence according to  claim 44 , wherein said polypeptide has increased resistance to proteolysis in comparison with that of the polypeptide whose sequence is shown in SEQ ID NO: 1.  
     
     
         90 . A nucleotide sequence according to  claim 44 , wherein the amino acid at position X is a hydrophobic amino acid.  
     
     
         91 . A nucleotide sequence according to  claim 44 , wherein the amino acid at position X is a charged amino acid.  
     
     
         92 . A nucleotide sequence according to  claim 91 , wherein the amino acid at position X is a negatively charged amino acid.  
     
     
         93 . A nucleotide sequence according to  claim 44  that encodes a modified EGF polypeptide having a length of 51 amino acids, wherein the modified EGF polypeptide has an amino acid sequence at least 75% identical to that of positions 1-47 of SEQ ID NO:1, wherein the modified EGF polypeptide has a biological activity that is at least 50% that of the polypeptide of SEQ ID NO: 1, wherein the activity is selected from the group consisting of mitogenesis, cell growth stimulation, tissue growth stimulation, cytoprotection, inhibition of acid secretion migration stimulation, wound healing, growth of a tissue precursor cell, differentiation of a tissue precursor cell, growth and differentiation of a tissue precursor cell, and EGF receptor binding, and wherein the amino acid residue at position 51 of the modified EGF polypeptide is an amino acid other than glutamic acid.  
     
     
         94 . A nucleotide sequence according to  claim 44  that encodes a polypeptide of 51 amino acids in length, wherein the amino acid sequence of residues 1-50 are at least 75% identical to the amino acid sequence as shown in SEQ ID NO:1, and residue 51 of the polypeptide is an asparagine residue, and wherein the polypeptide has a biological activity that is at least 50% that of the polypeptide whose sequence is shown in SEQ ID NO: 1, the activity being selected from the group consisting of mitogenesis, cell growth stimulation, tissue growth stimulation, cytoprotection, inhibition of acid secretion, migration stimulation, wound healing, growth of a tissue precursor cell, differentiation of a tissue precursor cell, growth and differentiation of a tissue precursor cell, and EGF receptor binding.  
     
     
         95 . A nucleotide sequence according to  claim 44  that encodes a human epidermal growth factor (EGF) polypeptide having an amino acid sequence at least 75% identical 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, wherein the polypeptide is more stable to proteolysis than a polypeptide having the amino acid sequence of SEQ ID NO: 1, the composition having a biological activity that is at least 50% that of the polypeptide whose sequence is shown in SEQ ID NO: 1, the activity being selected from the group consisting of mitogenesis, cell growth stimulation, tissue growth stimulation, cytoprotection, inhibition of acid secretion, migration stimulation, wound healing, growth of a tissue precursor cell, differentiation of a tissue precursor cell, growth and differentiation of a tissue precursor cell, and EGF receptor binding.  
     
     
         96 . The nucleotide sequence according to  claim 95 , wherein said amino acid sequence comprises an amino acid sequence substantially identical to the amino acid sequence of residues at positions 1-50 of SEQ ID NO: 1, and in which the residue at position 51 is an amino acid other than glutamic acid.  
     
     
         97 . The nucleotide sequence according to  claim 95 , wherein said residue at position 51 is selected from the group consisting of asparagine, glutamine, alanine, and serine.  
     
     
         98 . A nucleotide sequence according to  claim 44  encoding a modified epidermal growth factor polypeptide having biological activity of a wild-type human epidermal growth factor.  
     
     
         99 . A nucleotide sequence that encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3 and 4.  
     
     
         100 . The nucleotide sequence according to  claim 99 , wherein said polypeptide has increased resistance to proteolysis in comparison with that of the polypeptide whose sequence is shown in SEQ ID NO: 1.  
     
     
         101 . The nucleotide sequence according to  claim 99 , wherein at least one of residues at positions 1-50 is a conservative substitution of an amino acid in the sequence as shown in SEQ ID NO: 1.  
     
     
         102 . The nucleotide sequence according to  claim 99 , wherein said polypeptide further comprises a deletion of at least one of residues selected from amino acids at positions 1-5 as shown in SEQ ID NO: 1.