Production of recombinant lactoferrin and lactoferrin polypeptides using cDNA sequences in various organisms

Abstract

The verified cDNA sequences for human, bovine and porcine lactoferrin protein have been used to prepare recombinant lactoferrin for therapeutic and nutritional applications. Regions of the cDNA such as the Fe binding sites can be used to make an hLF polypeptide product The present invention provides novel plasmids, transfected eucaryotic cells and methods of producing these plasmids and transfected eucaryotic cells. The novel plasmid contains the cDNA for lactoferrin protein. Methods for the production of lactoferrin protein in fungi and bacteria are also provided. Thus, the present invention provides an efficient and economical means for the production of recombinant lactoferrin protein and lactoferrin related polypeptides.

1 - 23 . (Canceled)  
     
     
         24 . A process for producing lactoferrin which comprises culturing a transformant eucaryotic cell containing a recombinant plasmid, said plasmid comprising a plasmic vector having a polydeoxyribonucleotide which codes for a lactoferrin proteins in a suitable nutrient medium until the lactoferrin protein is formed and isolating the lactoferin protein.  
     
     
         25 - 29 . (Canceled)  
     
     
         30 . A method for producing biologically active recombinant lactoferrin comprising the steps of: 
 combining sequences containing a selectable marker gene, a promotor, a transcription termination sequence, and a linker sequence;    cloning said sequences to form a plasmid;    digesting said plasmid with a restriction endonuclease; 
 inserting a cDNA coding for human, bovine or porcine lactoferrin into a restriction site; and  
 transforming a cells with said plasmid to produce said recombinant lactoferrin.  
   
     
     
         31 . The method of  claim 30 , wherein said selectable marker gene is selected from the group consisting of pryr4, pyrG, andS, argB and trpC.  
     
     
         32 . Canceled  
     
     
         33 . The method of  claim 30 , wherein said promotor is selected from the group consisting of alcohol dehydrogenase, argB, α-amylase, glucoamylase, alcohol dehydrogenase and benA.  
     
     
         34 . The method of  claim 30 , wherein said transcription termination sequence is selected from the group consisting of α-amylase, glucoamylase, alcohol dehydrogenase and bena.  
     
     
         35 . The method of  claim 30 , wherein said linker sequence is selected from the group consisting of α-amylase, glucoamylase and lactoferrin.  
     
     
         36 - 57 . (Canceled)  
     
     
         58 . A method for producing biologically active recombinant lactoferrin comprising the steps of: 
 combining sequences containing a selectable marker gene, a promotor, a transcription termination sequence, and a linker sequence;    cloning said sequences to form a plasmid;    digesting said plasmid with a restriction endocnuclease;    inserting a substitution analog of a cDNA sequence selected from the group consisting of SEQ. ID No. into a restriction site; and transforming eucaryotic cells with said plasmid expressing lactoferrin cDNA which produces said recombinant lactoferrin.    
     
     
         59 . The method of  claim 58 , wherein said selectable marker gene is selected from the group consisting of pyr4, pyrG, andS, argB and trpC.  
     
     
         60 . Canceled  
     
     
         61 . A recombinant lactoferrin produced by the method of  claim 58 .  
     
     
         62 . The method of  claim 58 , wherein said promotor is selected from the group consisting of alcohol dehydrogenase, argB, α-amylase, glucoamylase, and benA.  
     
     
         63 . The method of  claim 58 , wherein said linker sequence is selected from the group consisting of α-amylase, glucoamylase, alcohol dehydrogenase and benA.  
     
     
         64 . The method of  claim 58 , wherein said linker sequence is selected from the group consisting of α-amylase, glucoamylase, and lactoferrin.  
     
     
         65 - 68 . Canceled.