Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof

Abstract

The invention features non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof.

1 . A nucleic acid molecule encoding non-glycosylated human alpha-fetoprotein (ng.HuAFP), or a nonglycosylated fragment thereof.  
     
     
         2 . The nucleic acid molecule of  claim 1 , comprising nucleotides 45 through 1874 of the sequence set forth in SEQ ID NO: 5.  
     
     
         3 . A nucleic acid molecule comprising: (i) a nucleic acid sequence encoding ng.HuAFP, (ii) a promoter that is operably linked to said ng.HuAFP-encoding sequence that enables expression of ng.HuAFP, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of said ng.HuAFP by a cell.  
     
     
         4 . The nucleic acid molecule of  claim 3 , wherein said cell is an  E. coli.    
     
     
         5 . The nucleic acid molecule of  claim 3 , wherein said cell is a eukaryotic cell.  
     
     
         6 . The nucleic acid molecule of  claim 5 , wherein said eukaryotic cell is a yeast cell or an animal cell.  
     
     
         7 . The nucleic acid molecule of  claim 6 , wherein said animal cell is in a transgenic animal.  
     
     
         8 . The nucleic acid molecule of  claim 7 , wherein said transgenic animal is a mammal.  
     
     
         9 . The nucleic acid molecule of  claim 8 , wherein said mammal is a goat, sheep, camel, cow, pig, rabbit, horse, or llama.  
     
     
         10 . The nucleic acid molecule of  claim 3 , wherein said cell is a biological fluid-producing cell in a transgenic animal; said promoter enables expression of said ng.HuAFP in said biological fluid-producing cell; and said leader sequence enables secretion of said ng.HuAFP into a biological fluid of said transgenic animal.  
     
     
         11 . The nucleic acid molecule of  claim 10 , wherein said biological fluid is milk, urine, blood, or lymph.  
     
     
         12 . The nucleic acid molecule of  claim 3 , wherein: said cell is in a transgenic animal; said promoter is a milk-specific promoter that enables expression of said ng.HuAFP in a milk-producing cell of said animal; and said leader sequence enables secretion of said ng.HuAFP into the milk of said animal.  
     
     
         13 . The nucleic acid molecule of  claim 3 , wherein: said cell is in a transgenic animal; said promoter is a urine-specific promoter that enables expression of said ng.HuAFP in a urine-producing cell of said animal; and said leader sequence enables secretion of said ng.HuAFP into the urine of said animal.  
     
     
         14 . The nucleic acid molecule of  claim 3 , wherein: said cell is in a transgenic animal; said promoter is a blood-specific promoter that enables expression of said ng.HuAFP in a blood-producing cell of said animal; and said leader sequence enables secretion of said ng.HuAFP into the blood of said animal.  
     
     
         15 . The nucleic acid molecule of  claim 3 , wherein said cell is in a transgenic animal; said promoter is a lymph-specific promoter that enables expression of said ng.HuAFP in a lymph-producing cell of said animal; and said leader sequence enables secretion of said ng.HuAFP into the lymph of said animal.  
     
     
         16 . A method of producing ng.HuAFP, said method comprising the steps of: 
 (a) providing a cell transduced with a transgene that comprises: (i) a nucleic acid molecule encoding n.g.HuAFP comprising nucleotides 45 through 1874 of the nucleic acid sequence set forth in SEQ ID NO: 5, (ii) a promoter that is operably linked to said n.g.HuAFP-encoding molecule and that enables expression of said n.g.HuAFP by said cell, and (iii) a leader sequence encoding a protein secretory signal that enables secretion of said n.g.HuAFP by said cell; and    (b) growing said transduced cell, wherein said cell expresses and secretes said ng.HuAFP.    
     
     
         17 . The method of  claim 16 , wherein said cell is an  E. coli.    
     
     
         18 . The method of  claim 16 , wherein said cell is a eukaryotic cell  
     
     
         19 . The method of  claim 18 , wherein said eukaryotic cell is a yeast cell or an animal cell.  
     
     
         20 . The method of  claim 19 , wherein said yeast cell is  Pichia pastoris.    
     
     
         21 . The method of  claim 17 , wherein said cell secretes said ng.HuAFP into cell culture medium.  
     
     
         22 . The method of  claim 19 , wherein said animal cell is a milk-producing, urine-producing, blood-producing, or lymph-producing cell.  
     
     
         23 . The method of  claim 18 , wherein said cell secretes said ng.HuAFP into cell culture medium.