Modified Cyanovirin-N Polypeptide

Abstract

This disclosure relates to a modified version of cyanovirin-N protein and a transformed Lactobacillus bacterium that is capable of recombinantly expressing this modified protein. Further disclosed are polynucleotide sequence encoding the modified cyanovirin-N protein, expression cassette that allows the expression of the modified protein, and method of make and use of the modified protein as well as the transformed Lactobacillus bacterium. In addition, this disclosure describes two novel promoters originated from L. jensenii , which are capable of directing a high level of gene expression in Lactobacillus bacteria. Also described are an expression cassette comprising one of the novel promoters, a genetically modified Lactobacillus bacterium containing the expression cassette, a method for recombinant gene expression in Lactobacillus bacteria using the novel promoters, and a method for delivery of proteins with desired biological activity to a mucosal surface in a human.

1 . A  Lactobacillus  bacterium expressing a modified polypeptide comprising a core amino acid sequence identical to the amino acid sequence of the unmodified polypeptide and an additional amino acid sequence immediately to the N-terminus of the core amino acid sequence, wherein the additional amino acid sequence consists of two to twenty amino acids, among which at least one of the first two amino acids is identical to its corresponding amino acid in the first two amino acids of a mature secreted  Lactobacillus  protein following the cleavage of a signal sequence, wherein the modified polypeptide is derived from a precursor polypeptide following the cleavage of the signal sequence of the secreted  Lactobacillus  protein, and wherein the modified polypeptide retains the same biological activity as the unmodified polypeptide. 
     
     
         2 . The bacterium of  claim 1 , wherein the additional amino acid sequence is selected from the group consisting of AP, APV, APVT, APAS, APVN, APVTNV, and SP. 
     
     
         3 . A modified polypeptide comprising a core amino acid sequence identical to the amino acid sequence of the unmodified polypeptide and an additional amino acid sequence immediately to the N-terminus of the core amino acid sequence, wherein the additional amino acid sequence consists of two to twenty amino acids, among which at least one of the first two amino acids is identical to its corresponding amino acid in the first two amino acids of a mature secreted  Lactobacillus  protein following the cleavage of a signal sequence, wherein the modified polypeptide is derived from a precursor polypeptide following the cleavage of the signal sequence of the secreted  Lactobacillus  protein, and wherein the modified polypeptide retains the same biological activity as the unmodified polypeptide. 
     
     
         4 . The modified polypeptide of  claim 3 , wherein the additional amino acid sequence is selected from the group consisting of AP, APV, APVT, APAS, APVN, APVTNV, and SP. 
     
     
         5 . A precursor polypeptide of the modified polypeptide of  claim 3 , comprising the signal sequence at the N-terminus of the modified polypeptide. 
     
     
         6 . A composition comprising the bacterium of  claim 1  and a physiologically acceptable carrier. 
     
     
         7 . A composition comprising the modified polypeptide of  claim 3  and a physiologically acceptable carrier. 
     
     
         8 . An isolated polynucleotide sequence encoding the modified polypeptide of  claim 3 . 
     
     
         9 . An isolated polynucleotide sequence encoding the precursor polypeptide of  claim 5 . 
     
     
         10 . An expression cassette comprising the polynucleotide sequence of  claim 9  operably linked to a promoter. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . A method for recombinantly producing a modified polypeptide, comprising the steps of introducing the polynucleotide sequence of  claim 9  into a suitable host cell and culturing the cell under conditions permitting the expression of the modified polypeptide. 
     
     
         14 - 19 . (canceled) 
     
     
         20 . The bacterium of  claim 1 , wherein the secreted  Lactobacillus  protein is CbsA protein. 
     
     
         21 . The modified polypeptide of  claim 3 , wherein the secreted  Lactobacillus  protein is CbsA protein. 
     
     
         22 . The precursor polypeptide of  claim 5 , wherein the secreted  Lactobacillus  protein is CbsA protein. 
     
     
         23 . The composition of  claim 6  or  7 , wherein the secreted  Lactobacillus  protein is CbsA protein. 
     
     
         24 . The polynucleotide sequence of  claim 8  or  9 , wherein the secreted  Lactobacillus  protein is CbsA protein. 
     
     
         25 . The expression cassette of  claim 10 , wherein the secreted  Lactobacillus  protein is CbsA protein.