Co-expression of recombinant proteins

Abstract

Expression vectors are described which permit the recombinant expression of proteins which essentially contain, in addition to nucleic acid encoding the recombinant protein, nucleic acid encoding a non-proteolytic analog of Haemophilus Hin47 protein, with or without leader sequence, or nucleic acid encoding high molecular weight proteins of non-typeable Haemophilus, which are hmwB, hmwC or hmwBC.

1 . An expression vector, comprising: 
 a nucleic acid molecule encoding a non-proteolytic analog of a Hin47 protein of a strain of  Haemophilus  including a portion thereof encoding the leader sequence for said non-proteolytic analog, and    a promoter operatively connected to said nucleic acid molecule to direct expression of said non-proteolytic analog of a Hin47 protein having said leader sequence in a host cell.    
     
     
         2 . The vector of  claim 1  wherein said non-proteolytic analog of Hin47 protein is a mutation of natural Hin47 protein in which at least one amino acid selected from the group consisting of amino acids 91, 121 and 195 to 201 of natural Hin47 protein has been deleted or replaced by a different amino acid.  
     
     
         3 . The vector of  claim 2  wherein histidine 91 is replaced by alanine.  
     
     
         4 . The vector of  claim 1  which is plasmid vector having the identifying characteristics of plasmid JB-3120-2 as seen in  FIG. 1A .  
     
     
         5 . An expression vector for expression of a recombinant protein in a host cell, comprising: 
 a nucleic acid molecule encoding a non-proteolytic analog of a  Haemophilus  Hin47 protein,    at least one additional nucleic acid molecule encoding the recombinant protein, and    at least one regulatory element operatively connected to said first nucleic acid molecule and said at least one additional nucleic acid molecule to effect expression of at least said recombinant protein in the host cell.    
     
     
         6 . The vector of  claim 5  wherein said nucleic acid molecule encoding the non-proteolytic analog of a Hin47 protein includes a portion encoding the leader sequence for said non-proteolytic analog.  
     
     
         7 . The vector of  claim 6  wherein said non-proteolytic analog of Hin47 protein is a mutation of natural Hin47 protein in which at least one amino acid selected from the group consisting of amino acids 91, 121 and 195 to 201 of natural Hin47 protein has been deleted or replaced by a different amino acid.  
     
     
         8 . The vector of  claim 7  wherein histidine 91 is replaced by alanine.  
     
     
         9 . The vector of  claim 5  wherein said at least one additional nucleic acid molecule encodes a Hia or Hsf protein of a strain of  Haemophilus influenzae.    
     
     
         10 . The vector of  claim 9  wherein said at least one additional nucleic acid molecule encodes a Hia protein which is N-terminally truncated.  
     
     
         11 . The vector of  claim 10  wherein said N-terminal truncation is S44 or V3 8.  
     
     
         12 . The vector of  claim 11  which is a plasmid vector having the identifying characteristics of plasmid DS-2342-2-2 as seen in  FIG. 5 .  
     
     
         13 . The vector of  claim 11  which is a plasmid vector having the identifying characteristics of plasmid JB-3145-1 seen in  FIG. 10 .  
     
     
         14 . The vector of  claim 5  wherein said at least one additional nucleic acid molecule encodes a PsaA protein of a strain of Streptococcus pneumoniae.  
     
     
         15 . The vector of  claim 14  which is a plasmid vector having the identifying characteristics of plasmid JB-3073R-1 as seen in  FIG. 12 .  
     
     
         16 . The vector of  claim 14  which is a plasmid vector having the identifying characteristics of plasmid JB-3090-1 or JB-3090-7 as seen in  FIG. 13 .  
     
     
         17 . A method for expressing at least one protein, which comprises: 
 providing a first nucleic acid molecule encoding a non-proteolytic analog of a Hin47 protein of  Haemophilus;      isolating at least one additional nucleic acid molecule encoding a protein other than Hin47;    introducing the first nucleic acid molecule and the at least one additional nucleic acid molecule into a cell to produce a transformed cell; and    growing the transformed cell to produce at least one protein.    
     
     
         18 . The method of  claim 17  wherein said first nucleic acid molecule contains a portion encoding the leader sequence for said non-proteolytic analog.  
     
     
         19 . The method of  claim 18  wherein said non-proteolytic analog of Hin47 protein is a mutation of natural Hin47 protein in which at least one amino acid selected from the group consisting of amino acids 91, 121 and 195 to 201 of natural Hin47 protein has been deleted or replaced by a different amino acid.  
     
     
         20 . The method of  claim 19  wherein histidine 91 is replaced by alanine.  
     
     
         21 . The method of  claim 20  wherein said at least one additional nucleic acid molecule encodes a Hia or Hsf protein of a strain of  Haemophilus influenzae.    
     
     
         22 . The method of  claim 21  wherein said at least one additional nucleic acid molecule encodes a Hia protein which is N-terminally truncated.  
     
     
         23 . The method of  claim 22  wherein said N-terminal truncation is S44 or V38.  
     
     
         24 . The method of  claim 21  wherein said vector is a plasmid vector having the identifying characteristics of plasmid JB-3145-1 as shown in  FIG. 10 .  
     
     
         25 . The method of  claim 20  wherein said at least one additional nucleic acid molecule encodes a PsaA protein of a strain of  Streptococcus pneumoniae.    
     
     
         26 . The method of  claim 17  wherein said first nucleic acid molecule encodes the nature form of the non-proteolytic analog.  
     
     
         27 . The method of  claim 26  wherein said non-proteolytic analog of Hin47 protein is a mutation of natural Hin47 protein in which at least one amino acid selected from the group consisting of amino acids 91, 121 and 195 to 201 of natural Hin47 protein has been deleted or replaced by a different amino acid.  
     
     
         28 . The method of  claim 27  wherein histidine 91 is replaced by alanine.  
     
     
         29 . The method of  claim 28  wherein said at least one additional nucleic acid molecule encodes a Hia or Hsf protein of a strain of  Haemophilus influenzae.    
     
     
         30 . The method of  claim 29  wherein said at least one additional nucleic acid molecule encodes a Hia protein which is N-terminally truncated.  
     
     
         31 . The method of  claim 30  wherein said N-terminal truncation is S44 or V38.  
     
     
         32 . The method of  claim 31  wherein said vector is a plasmid vector having the identifying characteristics of plasmid DS-2342-2-2 as seen in  FIG. 5 .  
     
     
         33 . The method of  claim 31  wherein said vector is a plasmid vector having the identifying characteristics of plasmid JB-3134-1-1 as seen in  FIG. 9 .  
     
     
         34 . The method of  claim 28  wherein said at least one additional nucleic acid molecule encodes a PsaA protein of a strain of  Streptococcus pneumoniae.    
     
     
         35 . The method of  claim 34  wherein said vector is a plasmid vector having the identifying characteristics of plasmid JB-3073R-1 as seen in  FIG. 12 .  
     
     
         36 . The method of  claim 34  wherein said vector is a plasmid vector having the identifying characteristics of plasmid JB-3090-1 or JB-3090-7 as seen in  FIG. 13 .  
     
     
         37 . An expression vector, comprising: 
 a nucleic acid molecule encoding a high molecular weight protein of a non-typeable strain of  Haemophilus selected  from the group consisting of hmwB and hmwC, and    a promoter operatively connected to said nucleic acid molecule to direct expression of said high molecular weight protein in a host cell.    
     
     
         38 . The vector of  claim 37  which is a plasmid vector having the identifying characteristics of plasmid IN-137-1-16 shown in  FIG. 18A .  
     
     
         39 . The vector of  claim 37  which is a plasmid vector having the identifying characteristics of pT7 hmwC shown in  FIG. 19A .  
     
     
         40 . An expression vector for expression of a recombinant protein in a host cell, comprising: 
 a nucleic acid molecule encoding a high molecular weight (HMW) protein of a non-typeable strain of  Haemophilus  selected from the group consisting of hmwBC, hmwB and hmwC,    at least one additional nucleic acid molecule encoding the recombinant protein, and    at least one regulatory element operatively connected to said first nucleic acid molecule and said at least one additional nucleic acid molecule to effect expression of at least said recombinant protein in the host cell.    
     
     
         41 . The expression vector of  claim 40  wherein said at least one additional nucleic acid molecule is inserted into a plasmid having the identifying characteristics of plasmid IN-52-1-13 as shown in  FIG. 17A  and under control of said at least one regulatory element.  
     
     
         42 . The expression vector of  claim 40  wherein said at least one additional nucleic acid molecule is inserted into a plasmid having the identifying characteristics of plasmid IN-137-1-16 shown in  FIG. 18A  and under control of said at least one regulatory element.  
     
     
         43 . The expression vector of  claim 40  wherein said at least one additional nucleic acid molecule is inserted into a plasmid having the identifying characteristics of plasmid pT7 hmwC shown in  FIG. 19A  and under control of said at least one regulatory element.