Flavobacterium heparinum expression system

Abstract

This invention is directed to Flavobacterium heparinum for use as a host cell organism for the expression of homologous and heterologous genes.

1 . A host cell for recombinant DNA expression comprising  Flavobacterium heparinum.    
     
     
         2 . The host cell of  claim 1  further comprising a vector.  
     
     
         3 . The host cell of  claim 2  wherein said vector is a plasmid system.  
     
     
         4 . The host cell of  claim 3  wherein said plasmid system is selected from the group consisting of a modified broad-host plasmid.  
     
     
         5 . The host cell of  claim 1  wherein said recombinant DNA is integrated into the  Flavobacterium heparinum  chromosome.  
     
     
         6 . The host cell of  claim 5  wherein said recombinant DNA is integrated through homologous recombination.  
     
     
         7 . The host cell of  claim 6  wherein a gene encoded by said integrated DNA is expressed at high levels.  
     
     
         8 . The host cell of  claim 5  wherein said recombinant DNA is integrated through any of bacteriophage integration, transposition of a transposon and transposition of an insertion sequence element.  
     
     
         9 . The host cell of  claim 1  further comprising a selective marker for selection of host cells expressing a desired recombinant DNA.  
     
     
         10 . The host cell of  claim 9  wherein said selective marker comprises one or more of a gene encoding antibiotic resistance, heavy metal resistance, a physiological growth inhibitory factor, and an amino acid requirement factor.  
     
     
         11 . The host cell of  claim 10  wherein said selective marker expression is regulated by a regulatory region from  Flavobacterium heparinum.    
     
     
         12 . The host cell of  claim 11  wherein said regulatory region is the heparinase I gene regulatory region.  
     
     
         13 . The host cell of  claim 1  wherein said recombinant DNA is expressed under the control of a regulatory region from  Flavobacterium heparinum.    
     
     
         14 . The host cell of  claim 13  wherein said regulatory region is the heparinase I gene regulatory region.  
     
     
         15 . The host cell of  claim 1  wherein said recombinant DNA is introduced into said cell by conjugation.  
     
     
         16 . The host cell of  claim 1  wherein said recombinant DNA is introduced into said cell by electroporation.  
     
     
         17 . The host cell of  claim 1  wherein said recombinant DNA is introduced into said cell by bacterial phage transfection.  
     
     
         18 . The host cell of  claim 1  wherein said cell glycosylates glycoproteins encoded by said recombinant DNA.  
     
     
         19 . The host cell of  claim 1  wherein said cell expresses recombinant DNA containing a homologous gene.  
     
     
         20 . The host cell of  claim 1  wherein said cell expresses recombinant DNA containing a heterologous gene.  
     
     
         21 . A  Flavobacterium heparinum  host organism transformed with recombinant DNA comprising a homologous or a heterologous gene placed under the control of a gene promoter derived from a protein endogenous to the  F. heparinum  host and operably linked to the coding sequence for the homologous or heterologous gene.  
     
     
         22 . The  F. heparinum  host organism of  claim 21 , wherein said gene promoter is hepA.  
     
     
         23 . A method for producing a desired polypeptide or protein comprising expressing recombinant DNA comprising a coding sequence for the desired polypeptide or protein in a  F. heparinum  host organism;  
     
     
         24 . The method of  claim 23 , wherein the expressed polypeptide or protein is glycosylated.  
     
     
         25 . The method of  claim 23 , wherein the expressed polypeptide or protein is biologically active.  
     
     
         26 . An expression system for expressing a desired polypeptide or protein comprising: 
 (1) a  F. heparinum  host organism    (2) nucleotide sequences encoding a desired polypeptide or protein, and    (3) a vector for expressing the nucleotide sequences capable of expressing the desired polypeptide or protein.    
     
     
         27 . A vector which upon introduction into a  F. heparinum  host cell effects expression of DNA encoding a desired polypeptide or protein, the vector comprising (a) a functional origin of replication (OriC) region; (b) replication (rep) genes; and (c) a gene promoter derived from a protein endogenous to the  F. heparinum  host.  
     
     
         28 . The vector depicted in  FIG. 1 .  
     
     
         29 . The vector depicted in  FIG. 2 .