Lipid A 4' kinase

Abstract

The present invention relates, in general, to lipid A 4′ kinase and, in particular, to a nucleic acid encoding lipid A 4′ kinase and to a method of producing lipid A 4′ kinase recombinantly using same. The invention further relates to methods of producing 4′ phosphorylated lipid A analogs using the recombinantly produced lipid A 4′ kinase.

What is claimed is:  
     
         1 . An isolated nucleic acid comprising a sequence encoding lipid A 4′ kinase, said nucleic acid being free of an encoding sequence with which said lipid A 4′ kinase encoding sequence is naturally associated within an operon.  
     
     
         2 . The nucleic acid according to  claim 1  wherein said lipid A 4′ kinase is  E. coli  lipid A 4′ kinase.  
     
     
         3 . The nucleic acid according to  claim 1  wherein said lipid A 4′ kinase has the amino acid sequence set forth in FIG. 1B.  
     
     
         4 . An isolated nucleic acid encoding lipid A 4′ kinase, or portion thereof of at least 18 consecutive bases, or complement thereof, said nucleic acid being free of an encoding sequence with which said lipid A 4′ kinase encoding sequence is naturally associated within an operon.  
     
     
         5 . The nucleic acid according to  claim 4  wherein said lipid A 4′ kinase is  E. coli  lipid A 4′ kinase.  
     
     
         6 . The isolated nucleic acid according to  claim 5  wherein the nucleic acid encodes the amino acid sequence set forth in FIG. 1B, or portion thereof of at least 6 amino acids.  
     
     
         7 . The isolated nucleic acid according to  claim 6  wherein the nucleic acid has the sequence shown in FIG. 1A, or variant thereof, or portion thereof of at least 18 consecutive bases.  
     
     
         8 . The isolated nucleic acid according to  claim 7  wherein said nucleic acid has the sequence shown in FIG. 1A, or portion thereof of at least 18 consecutive bases.  
     
     
         9 . The isolated nucleic acid according to  claim 8  wherein the nucleic acid has the sequence shown in FIG. 1A.  
     
     
         10 . A recombinant molecule comprising said nucleic acid according to  claim 1  and a vector.  
     
     
         11 . The recombinant molecule according to  claim 10  further comprising a promoter operably linked to said nucleic acid sequence.  
     
     
         12 . A recombinant molecule comprising a nucleic acid encoding lipid A 4′ kinase operably linked to a heterologous regulatory sequence.  
     
     
         13 . A host cell comprising said recombinant molecule according to  claim 10 .  
     
     
         14 . A host cell comprising said recombinant molecule according to  claim 12 .  
     
     
         15 . A method of producing lipid A 4′ kinase comprising culturing said host cell according to  claim 13  under conditions such that said nucleic acid sequence is expressed and said lipid A 4′ kinase is thereby produced.  
     
     
         16 . A method of producing lipid A 4′ kinase comprising culturing said host cell according to  claim 14  under conditions such that said nucleic acid sequence is expressed and said lipid A 4′ kinase is thereby produced.  
     
     
         17 . A recombinant molecule comprising the nucleic acid according to  claim 4  and a vector.  
     
     
         18 . The recombinant molecule according to  claim 17  further comprising a promoter operably linked to said nucleic acid.  
     
     
         19 . A host cell comprising said recombinant molecule according to  claim 17 .  
     
     
         20 . A method of producing lipid-A 4′ kinase or portion thereof, comprising culturing said host cell according to  claim 19  under conditions such that said nucleic acid sequence is expressed and said lipid A 4′ kinase, or portion thereof, is thereby produced.  
     
     
         21 . An isolated lipid A 4′ kinase or portion thereof of at least 6 consecutive amino acids, wherein said lipid A 4′ kinase, or portion thereof, is free of proteins with which said lipid A 4′ kinase is naturally expressed from a common promoter.  
     
     
         22 . The lipid A 4′ kinase according to  claim 21  wherein said lipid A 4′ kinase is free of msbA.  
     
     
         23 . The lipid A 4′ kinase according to  claim 21  wherein said lipid A 4′ kinase has the amino acid sequence shown in FIG. 1B.  
     
     
         24 . A fusion product comprising the lipid A 4′ kinase or portion thereof of  claim 21  and non-lipid A 4′ kinase amino acid sequence.  
     
     
         25 . A composition comprising lipid A 4′ kinase wherein when said composition further comprises a protein expressed from a promoter from which said lipid A 4′ kinase is naturally expressed, said lipid A 4′ kinase is present in said composition at a ratio of at least 100:1 relative to said protein as determined by quantitative Western blotting.  
     
     
         26 . An antibody specific for the lipid A 4′ kinase, or portion thereof, of  claim 21 , or binding fragment thereof.  
     
     
         27 . A method of treating a patient in need of immunostimulation comprising introducing into immune cells of said patient a construct comprising a nucleic acid encoding lipid A 4′ kinase, under conditions such that said nucleic acid is expressed and lipid A 4′ kinase is thereby produced, and administering to said patient a precursor of a lipid A agonist that requires phosphorylation for activation, wherein said administration is effected under conditions such that said lipid A 4′ kinase phosphorylates, and thereby activates, said precursor so that said treatment is effected.  
     
     
         28 . The method according to  claim 27  wherein said precursor is a disaccharide that requires 4′ phosphorylation for activation.  
     
     
         29 . A method of phosphorylating a precursor of a lipid A agonist or antagonist comprising contacting said precursor with said lipid A 4′ kinase according to  claim 21  under conditions such that said precursor is phosphorylated.  
     
     
         30 . The method according to  claim 29  wherein said precursor is a disaccharide and said phosphorylation occurs at the 4′ position.  
     
     
         31 . A method of phosphorylating a precursor of a lipid A agonist or antagonist comprising contacting said precursor with said composition according to  claim 24  under conditions such that said precursor is phosphorylated.  
     
     
         32 . The method according to  claim 31  wherein said precursor is a disaccharide and said phosphorylation occurs at the 4′ position.  
     
     
         33 . A method of phosphorylating a precursor of a lipid A agonist or antagonist comprising contacting said precursor with recombinant lipid A 4′ kinase under conditions such that said precursor is phosphorylated.  
     
     
         34 . The method according to  claim 33  wherein said precursor is a disaccharide and said phosphorylation occurs at the 4′ position.