Recombinant human mannan-binding lectin

Abstract

The present invention relates to methods of producing and/or isolating MBL compositions. In particular the invention relates to methods of producing and/or isolating novel recombinant human MBL preparations with high similarity to natural human MBL. The MBL preparations may be used in compositions, medicaments and in methods for treatment of conditions such as those related to immunosuppressive conditions and/or to conditions of MBL deficiencies including latent conditions. Conditions of deficiencies of Mannan-Binding Lectin (MBL) are associated with increased susceptibility to infections.

1 . A gene expression construct comprising a nucleotide sequence encoding a human mannan-binding lectin (MBL) polypeptide, 
 said nucleotide sequence comprising at least one intron sequence from the human MBL gene,    said construct comprising a promoter, different from the human MBL promoter, and operably linked to said nucleotide sequence in such manner that said polypeptide, or a precursor thereof, is expressed.    
     
     
         2 . The gene expression construct according to  claim 1 , wherein the promoter is a promoter of a eukaryotic gene.  
     
     
         3 . The gene expression construct according to  claim 2 , wherein the promoter is a promoter of a gene of a mammal or insect.  
     
     
         4 . The gene expression construct according to  claim 1 , wherein the promoter is a promoter of a viral gene.  
     
     
         5 . The gene expression construct according to  claim 1 , wherein the promoter is selected from the group consisting of Rous sarcoma virus long terminal repeat promoter, cytomegalovirus immediate-early promoter, and elongation factor-1 alpha promoter.  
     
     
         6 . The gene expression construct according to  claim 1 , wherein the promoter is a promoter of a microbial gene.  
     
     
         7 . The gene expression construct according to  claim 6 , wherein the promoter is a promoter of a gene of a virus, yeast or bacterium.  
     
     
         8 . The gene expression construct according to  claim 1 , wherein the construct comprises an enhancer element operably linked to said promoter.  
     
     
         9 . A vector comprising the gene expression construct according to  claim 1 , wherein the vector otherwise possesses the identifying characteristics of a pREP9 vector.  
     
     
         10 . The gene expression construct according to  claim 1  wherein the amino acid sequence of the encoded MBL is SEQ ID NO:1.  
     
     
         11 . A process of producing a human recombinant mannan binding lectin (MBL) having a ratio of (a) MBL tetramers, pentamers, and hexamers to (b) monomers and dimers higher than the ratio of (a) to (b) when produced recombinantly by using cDNA, comprising the step of: 
 cultivating a host cell culture transformed with an intron-containing gene expression construct according to  claim 1 , under conditions conducive to expression of said polypeptide or precursor thereof, and secretion of said polypeptide, thereby obtaining expression of said polypeptide or precursor thereof and secretion of the polypeptide into the culture medium, thereby producing, in said culture medium, human recombinant MBL, the latter having a ratio of (a) MBL tetramers, pentamers, and hexamers to (b) monomers and dimers which is higher than the ratio of (a) to (b) when produced recombinantly using a cDNA expression construct instead of said intron-containing gene expression construct, the constructs and cultivation conditions otherwise being essentially identical.    
     
     
         12 . The process according to  claim 11 , in which the DNA sequences encode a polypeptide sequence as shown in SEQ ID NO: 1.  
     
     
         13 . The process according to  claim 11 , wherein the host cell culture is cultured in vitro.  
     
     
         14 . The process according to  claim 11 , wherein the host cell culture is cultured in vivo.  
     
     
         15 . The process according to  claim 14 , wherein the host cell culture is cultured in a transgenic animal.  
     
     
         16 . The process according to  claim 11 , wherein the host cell culture is a eukaryotic host cell culture.  
     
     
         17 . The process according to  claim 16 , wherein the host cell culture is a mammalian host cell culture.  
     
     
         18 . The process according to  claim 11 , wherein the promoter is a promoter of a eukaryotic gene.  
     
     
         19 . The process according to  claim 18 , wherein the promoter is a promoter of a gene of a mammal or insect.  
     
     
         20 . The process according to  claim 11 , wherein the promoter is a promoter of a viral gene.  
     
     
         21 . The process according to  claim 11 , wherein the promoter is selected from the group consisting of Rous sarcoma virus long terminal repeat promoter, and cytomegalovirus immediate-early promoter, and elongation factor-1 alpha promoter.  
     
     
         22 . The process according to  claim 11 , wherein the promoter is a promoter of a microbial gene.  
     
     
         23 . The process according to  claim 22 , wherein the promoter is a promoter of a gene of a virus, yeast or bacterium.  
     
     
         24 . The process according to  claim 11 , wherein the construct comprises an enhancer element operably linked to said promoter.  
     
     
         25 . The process according to  claim 11 , wherein the host cell is transformed with a vector comprising said intron-containing construct, and the vector otherwise possesses the identifying characteristics of a pREP9 vector.