Expression of lipoproteins

Abstract

Heterologous lipidated proteins formed recombinantly are disclosed and claimed. The expression system can be E. coli. The heterologous lipidated protein has a leader sequence which does not naturally occur with the protein portion of the lipidated protein. The lipidated protein can have the Borrelia OspA leader sequence. The protein portion can be OspC, PspA, UreA, Ure B, or a fragment thereof. Methods and compositions for forming and employing the proteins are also disclosed and claimed.

1 . A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first-nucleic acid sequence, said first nucleic acid sequence being contiguous with said second nucleic acid sequence when the mature protein is naturally lipidated, or said first and second nucleic acid sequences being separated by one codon coding for one amino acid when the mature protein is not naturally lipidated.  
     
     
         2 . The hybrid nucleic acid molecule of  claim 1  wherein said signal sequence is the signal sequence of an OspA protein of a  Borrelia  species.  
     
     
         3 . The hybrid nucleic acid molecule of  claim 2  wherein said first nucleic acid sequence and said second nucleic acid sequence are coupled in a translational open reading frame relationship.  
     
     
         4 . The hybrid nucleic acid molecule of  claim 3  wherein said mature protein is an OspC lipoprotein of a  Borrelia  species, or a fragment thereof.  
     
     
         5 . The hybrid molecule of  claim 4  wherein said ospC lipoprotein is that of a strain of  B. burgdorferi.    
     
     
         6 . The hybrid molecule of  claim 5  wherein said strain of  B. burgdorferi  is selected from the OspC sub-type families.  
     
     
         7 . The hybrid molecule of  claim 5  wherein said OspA protein is that of a strain of  B. burgdorferi.    
     
     
         8 . The hybrid molecule of  claim 7  wherein said strain of  B. burgdorferi  is selected from the B31, ACA1 and Ip90 families of strains.  
     
     
         9 . The hybrid nucleic acid molecule of  claim 3  wherein said mature protein is a PspA protein of a strain of  S. pneumoniae,  or a fragment thereof.  
     
     
         10 . The hybrid molecule of  claim 9  wherein said OspA protein is that of a strain of  B. burgdorferi.    
     
     
         11 . The hybrid molecule of  claim 10  wherein said strain of  B. burgdorferi  is selected from the B31, ACA1 and Ip90 families of strains.  
     
     
         12 . The hybrid nucleic acid molecule of  claim 3  wherein said mature protein is a UreA protein of a strain of  H. pylori,  or a fragment thereof.  
     
     
         13 . The hybrid molecule of  claim 12  wherein said ospA protein is that of a strain of  B. burgdorferi.    
     
     
         14 . The hybrid molecule of  claim 13  wherein said strain of  B. burgdorferi  is selected from the B31, ACA1 and Ip90 families of strains.  
     
     
         15 . The hybrid nucleic acid molecule of  claim 3  wherein said mature protein is a UreB protein of a strain of  H. pylori,  or a fragment thereof.  
     
     
         16 . The hybrid molecule of  claim 15  wherein said OspA protein is that of a strain of  B. burgdorferi.    
     
     
         17 . The hybrid molecule of  claim 16  wherein said strain of  B. burgdorferi  is selected from the B31, ACA1 and Ip90 families of strains.  
     
     
         18 . A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding an OspC lipoprotein of a  Borrelia  species and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterologous to OspC and coupled in translational open reading frame relationship with said first nucleic acid sequence.  
     
     
         19 . The hybrid nucleic acid molecule of  claim 18  wherein said OspC lipoprotein is that of a strain of  B. burgdorferi.    
     
     
         20 . The hybrid nucleic acid molecule of  claim 19  wherein said strain of  B. burgdorferi  is selected from the OspC sub-type families.  
     
     
         21 . A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a PspA protein of a strain of  S. pneumoniae  and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to PspA and coupled in translational open reading frame relationship with said first nucleic acid sequence.  
     
     
         22 . A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a UreA protein of a strain of  H. pylori  and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to UreA and coupled in translational open reading frame relationship with said first nucleic acid sequence.  
     
     
         23 . A hybrid nucleic acid molecule, comprising a first nucleic acid sequence encoding a UreB protein of a strain of  H. pylori  and a second nucleic acid sequence encoding a signal sequence of an expressed protein heterlogous to UreB and coupled in translational open reading frame relationship with said first nucleic acid sequence.  
     
     
         24 . An expression vector containing the hybrid nucleic acid molecule of  claim 1  under control of a promoter for expression of said mature protein.  
     
     
         25 . The expression vector of  claim 24  wherein said mature protein is an OspC lipoprotein of a  Borrelia  species.  
     
     
         26 . The expression vector of  claim 24  wherein said mature protein is a PspA lipoprotein of a strain of  S. pneumoniae.    
     
     
         27 . The expression vector of  claim 24  wherein said mature protein is a UreA protein of a strain of  H. pylori.    
     
     
         28 . The expression vector of  claim 24  wherein said mature protein is a UreB protein of a strain of  H. pylori.    
     
     
         29 . An expression vector containing the hybrid nucleic acid molecule of  claim 18  under control of a promoter for expression of said OspC lipoprotein.  
     
     
         30 . An expression vector containing the hybrid nucleic acid molecule of  claim 21  under control of a promoter for expression of said PspA protein.  
     
     
         31 . An expression vector containing the hybrid nucleic acid molecule of  claim 22  under control of a promoter for expression of said UreA protein.  
     
     
         32 . An expression vector containing the hybrid nucleic acid molecule of  claim 23  under control of a promoter for expression of said UreB protein.  
     
     
         33 . A method for forming a recombinant protein, which comprises: 
 incorporating the expression vector of  claim 24  into a host organism; and    effecting expression of said mature protein from the host organism.    
     
     
         34 . The method of  claim 33  wherein said mature protein is an OspC lipoprotein of a  Borrelia  species.  
     
     
         35 . The method of  claim 34  wherein said host organism is  E. coli.    
     
     
         36 . The method of  claim 33  wherein said mature protein is a PspA protein of a strain of  S. pneumoniae.    
     
     
         37 . The method of  claim 36  wherein said host organism is  E. coli.    
     
     
         38 . The method of  claim 33  wherein said mature protein is a UreA protein of a strain of  H. pylori.    
     
     
         39 . The method of  claim 38 , wherein said host organism is  E. coli.    
     
     
         40 . The method of  claim 33  wherein said mature protein is a UreB protein of a strain of  H. pylori.    
     
     
         41 . The method of  claim 40  wherein said host organism is  E. coli.    
     
     
         42 . A method for forming recombinant ospC lipoprotein, which comprises: 
 incorporating the expression vector of  claim 29  into a host organism; and    effecting expression of said OspC lipoprotein from the host organism.    
     
     
         43 . The method of  claim 42  wherein said host organism is  E. coli.    
     
     
         44 . A method for forming recombinant PspA lipoprotein, which comprises: 
 incorporating the expression vector of  claim 30  into a host organism; and    effecting expression of said PspA lipoprotein from the host organism.    
     
     
         45 . The method of  claim 44  wherein said host organism is  E. coli.    
     
     
         46 . A method for forming recombinant UreA lipoprotein, which comprises: 
 incorporating the expression vector of  claim 31  into a host organism; and    effecting expression of said UreA lipoprotein from the host organism.    
     
     
         47 . The method of  claim 46  wherein said host organism is  E. coli.    
     
     
         48 . A method for forming recombinant UreB lipoprotein, which comprises: 
 incorporating the expression vector of  claim 32  into a host organism; and    effecting expression of said UreA lipoprotein from the host organism.    
     
     
         49 . The method of  claim 48  wherein said host organism is  E. coli.    
     
     
         50 . A process for the production of a recombinant lipoprotein, which comprises: 
 constructing a hybrid nucleic acid molecule comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first nucleic acid, said second nucleic acid sequence being contiguous with said first sequence;    forming an expression vector containing said hybrid nucleic acid molecule under control of a promoter for expression of said mature protein;    incorporating said expression vector into a host organism;    effecting expression of said recombinant lipoprotein by said host organism;    lysing the cells of the host organism;    treating the lysed cells with a surfactant which selectively solubilizes said recombinant lipoprotein in preference to bacterial and other proteins and which is able to effect phase separation of a detergent phase under mild conditions;    effecting phase separation at a detergent phase containing solubilized recombinant lipoprotein, an aqueous phase containing bacterial and other proteins and a solid phase containing cell residue;    separating and recovering said detergent phase from said solid phase and said aqueous phase;    contacting said detergent phase with a first chromatographic column under conditions which result in binding of protein other than said recombinant lipoprotein to said column to provide a flow-through from said first chromatographic column containing the recombinant lipoprotein and recovering said flow-through from said first chromatographic column;    contacting the flow-through from said first chromatographic column with a second chromatographic column under conditions which result in binding of the recombinant lipoprotein to the second chromatographic column in preference to contaminant proteins and lipopolysaccharides which pass through said second chromatographic column;    eluting said recombinant lipoprotein from said second chromatographic column to provide an eluant containing said recombinant lipoprotein substantially free from lipopolysaccharide and contaminant proteins; and    recovering said eluant.    
     
     
         51 . The process of  claim 50  wherein said signal sequence is the signal sequence of an OspA protein of a  Borrelia  species.  
     
     
         52 . The process of  claim 51  wherein said first nucleic acid sequence and said second nucleic acid sequence are coupled in a translational open reading frame relationship.  
     
     
         53 . The process of  claim 52  wherein said surfactant is TRITON™ X-114.  
     
     
         54 . The process of  claim 53  wherein said treating of lysed cells is effected at a temperature of about 0° C. to about 10° C., the resulting mixture is treated to a mildly elevated temperature of about 35° C. to about 40° C. to effect separation of said detergent phase, and said detergent phase is separated from said aqueous phase and said solid phase by centrifugation.  
     
     
         55 . The process of  claim 52  wherein said mature protein is an OspC lipoprotein of a  Borrelia  species.  
     
     
         56 . The process of  claim 55  wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant ospC lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column.  
     
     
         57 . The process of  claim 52  wherein said mature protein is a PspA protein of a strain of  S. pneumoniae.    
     
     
         58 . The process of  claim 57 , wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant PspA lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column.  
     
     
         59 . The process of  claim 52  wherein said mature protein is a UreA protein of a strain of  H. pylori.    
     
     
         60 . The process of  claim 59  wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant UreA lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column.  
     
     
         61 . The process of  claim 52  wherein said mature protein is a UreB protein of a strain of  H. pylori.    
     
     
         62 . The process of  claim 61  wherein said first chromatographic column is further contacted with a buffer medium at a pH to provide liquid containing the recombinant UreB lipoprotein from the first chromatographic column while the other proteins are retained on the first chromatographic column and the flow-through from the further contact is collected and combined with that from the first contacting step on said first chromatographic column and the combined flow-through is contacted with said second chromatographic column.  
     
     
         63 . The process of  claim 52  wherein said host organism lysis is effected by freezing and thawing the host organism.  
     
     
         64 . Recombinantly-produced, isolated and purified lipoprotein produced by the process of  claim 50 .  
     
     
         65 . Recombinantly-produced, isolated and purified ospC lipoprotein of a  Borrelia strain having a purity of at least about  80% and substantially free from contaminant proteins and lipopolysaccharides.  
     
     
         66 . Recombinantly-produced, isolated and purified lipidated PspA protein of a strain of  S. pneumoniae  having a purity of at least about 50% and substantially free from contaminant proteins and lipopblysaccharides.  
     
     
         67 . Recombinantly-produced, isolated and purified lipidated UreA protein of a strain of  H. pylori  having a purity of at least about 80% and substantially free from contaminant proteins and lipopolysaccharides.  
     
     
         68 . Recombinantly-produced, isolated and purified lipidated UreB protein of a strain of  H. pylori  having a purity of at least about 80% and substantially free from contaminant proteins and lipopolysaccharides.  
     
     
         69 . A lipoprotein fusion vector PLF100 having ATCC Accession No. 69750.  
     
     
         70 . A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipoprotein of  claim 64  or  65 .  
     
     
         71 . A composition for inducing an immunological response comprising the lipoprotein of  claim 64  or  65 .  
     
     
         72 . A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated PspA protein of  claim 64  or  66 .  
     
     
         73 . A composition for inducing an immunological response comprising the lipidated PspA protein of  claim 64  or  66 .  
     
     
         74 . A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated UreA protein of  claim 67 .  
     
     
         75 . A composition for inducing an immunological response comprising the lipidated UreA protein of  claim 67 .  
     
     
         76 . A method for inducing an immunological response in a human or animal comprising the step of administering to said human or animal a composition comprising the lipidated UreB protein of  claim 68 .  
     
     
         77 . A composition for inducing an immunological response comprising the lipidated UreB protein of  claim 68 .  
     
     
         78 . A method for enhancing the immunogenicity of a protein, comprising the steps of: 
 forming a hybrid nucleic acid molecule comprising a first nucleic acid sequence encoding a signal sequence of a lipoprotein and a second nucleic acid sequence encoding a mature protein, or fragment thereof, which is heterologous to the lipoprotein encoded by said first nucleic acid, said first nucleic acid sequence being contiguous with said second nucleic acid sequence;    incorporating said hybrid nucleic acid molecule into an expression vector;    effecting expression vector into a host organism; and    effecting expression of said mature protein from said host organism.