Kunitz domain polypeptide zkun10

Abstract

Proteinase inhibitors comprising a Kunitz domain are disclosed. The Kunitz domain comprises a motif of amino acid residues as shown in SEQ ID NO:4, and the sequence of the Kunitz domain is shown in residues 57 through 107 of SEQ ID NO:2. The polypeptide also includes an N-terminal collagen domain in which a von Willebrand domain resides, and is shown in SEQ ID NO: 5. Also disclosed are methods for making the proteinase inhibitors, and expression vectors and cultured cells that are useful within the methods. The proteinase inhibitors may be used as components of cell culture media, in protein purification, and as inhibitors of protease degradation of plasma proteins.

We claim:  
     
         1 . An isolated polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from residue 57 (Cys) to residue 107 (Cys).  
     
     
         2 . An isolated polypeptide comprising a sequence of amino acid residues with at least 95% identity to SEQ ID NO: 2 from residue 1 (Ile) to residue 111 (Gln).  
     
     
         3 . The polypeptide of  claim 2 , wherein any variation in the polypeptide in the region of amino acid residues 57 to 107 is subject to the limitations as shown in SEQ ID NO: 4.  
     
     
         4 . An isolated polypeptide comprising a sequence of amino acid residues with at least 95% identity to SEQ ID NO: 6 from residue 1 (Met) to residue 415 (Gln).  
     
     
         5 . The polypeptide of  claim 4 , wherein any variation in the polypeptide in the region of amino acid residues 361 (Cys) to 411 (Cys) is subject to the limitations as shown in SEQ ID NO: 4.  
     
     
         6 . An isolated polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 6 from amino acid residue 1 (Met) to amino acid residue 415 (Gln).  
     
     
         7 . A fusion protein comprising at least two polypeptides, wherein a first polypeptide and second polypeptide, and wherein at least one of the polypeptides comprise a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 57 (Cys) to amino acid residue 107 (Cys).  
     
     
         8 . A fusion protein comprising at least three polypeptides, wherein a first polypeptide comprises a secretory signal polypeptide followed by a second polypeptide comprising a collagen domain polypeptide containing one or more von Willebrand domains, followed by third polypeptide comprising one or more Kunitz domains, and wherein at least one of the Kunitz domains comprise a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 57 (Cys) to amino acid residue 107 (Cys).  
     
     
         9 . The fusion protein of  claim 8 , wherein the collagen domain further comprises at least one globular domains.  
     
     
         10 . An expression vector comprising the following operably linked elements: 
 (a) a transcription promoter;    (b) a DNA segment encoding a polypeptide according to  claim 1;  and    (c) a transcription terminator.    
     
     
         11 . The expression vector of  claim 10  further comprising a secretory signal sequence operably linked to the DNA segment.  
     
     
         12 . A cultured cell comprising the expression vector of  claim 10 .  
     
     
         13 . A method of making a polypeptide comprising: 
 culturing a cell according to  claim 12  under conditions wherein the DNA segment is expressed; and    recovering the protein encoded by the DNA segment.    
     
     
         14 . An antibody that specifically binds to the protein of  claim 1 .  
     
     
         15 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from residue 57 (Cys) to residue 107 (Cys).  
     
     
         16 . An isolated polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide comprising a sequence of amino acid residues with at least 95% identity to SEQ ID NO: 2 from residue 1 (Ile) to residue 111 (Gln).  
     
     
         17 . The polynucleotide molecule of  claim 16 , wherein any variation in the nucleotide sequence encoding a polypeptide that falls within the region of amino acid residues 57 to 107 is subject to the limitations as shown in SEQ ID NO: 4 for that corresponding region.  
     
     
         18 . An isolated polynucleotide molecule encoding a polypeptide comprising a sequence of amino acid residues with at least 95% identity to SEQ ID NO: 6 from residue 1 (Met) to residue 415 (Gln).  
     
     
         19 . The polynucleotide molecule of  claim 18 , wherein any variation in the nucleotide sequence encoding a polypeptide that falls within the region of amino acid residues 361 (Cys) to 411 (Cys) is subject to the limitations as shown in SEQ ID NO: 4 for that corresponding region.  
     
     
         20 . An isolated polynucleotide molecule comprising a sequence of polynucleotides selected from the group consisting of: 
 (a) a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 169 to nucleotide 321;    (b) a nucleotide sequence as shown SEQ ID NO: 1 from nucleotide 1 to nucleotide 333;    (c) a nucleotide sequence that encodes for a polypeptide as shown in SEQ ID NO: 2 from amino acid residue 57 to amino acid residue 107;    (d) a nucleotide sequence that encodes for a polypeptide as shown SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 111;    (e) a nucleotide sequence as shown SEQ ID NO: 5 from nucleotide 1 to nucleotide 1248; and    (f) a nucleotide sequence that encodes for a polypeptide as shown in SEQ ID NO: 6 from amino acid residue 1 to amino acid residue 415.    
     
     
         21 . A method of inhibiting protease degradation or activity in a composition containing plasma proteins comprising adding a zkun10 polypeptide composition comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 57 to amino acid residue 107 to the composition containing plasma proteins in an amount sufficient to reduce degradation of the composition by proteases or protease activity in the composition.  
     
     
         22 . The method of  claim 21 , wherein reduction of degradation or activity is determined by chromogenic substrate assays or clotting time assays.