Production of Biologically Active Proteins

Abstract

A fusion protein that is expressed in a recombinant protein body-like assembly (RPBLA) in host eukaryotic cells and organisms is disclosed. More particularly, a biologically active polypeptide fused to a protein sequence that mediates the induction of RPBLA formation is expressed and accumulated in host cells after transformation with an appropriate vector. The eukaryotic host cell does not produce protein bodies in the absence of the fusion protein. Methods for preparing and using the RPBLAs and the fusion protein are also disclosed, as are nucleic acid molecules that encode the fusion proteins.

1 - 15 . (canceled) 
     
     
         16 . A recombinant protein body-like assembly (RPBLA) that comprises a membrane-enclosed fusion protein, said fusion protein comprising two sequences linked together in which one sequence is a protein body-inducing sequence (PBIS) and the other is a biologically active polypeptide. 
     
     
         17 - 26 . (canceled) 
     
     
         27 . A vaccine or inoculum comprising an immunogenic effective amount of recombinant protein body-like assemblies (RPBLAs) that comprise a recombinant fusion protein dissolved or dispersed in a pharmaceutically acceptable diluent, said recombinant fusion protein comprising two sequences linked together in which one sequence is a protein body-inducing sequence (PBIS) and the other is an immunogenic polypeptide to which an immunological response is to be induced by said vaccine or inoculum. 
     
     
         28 . The vaccine or inoculum according to  claim 27  wherein said fusion protein further includes a linker sequence between the protein body-inducing sequence and the sequence of the immunogenic polypeptide. 
     
     
         29 . The vaccine or inoculum according to  claim 27  wherein the PBIS comprises a prolamin sequence. 
     
     
         30 . The vaccine or inoculum according to  claim 29  wherein the prolamin sequence is gamma-zein, alpha-zein, gamma-gliadin, or rice prolamin. 
     
     
         31 . The vaccine or inoculum according to  claim 30  wherein the prolamin sequence is the gamma-zein RX3 sequence. 
     
     
         32 . The vaccine or inoculum according to  claim 27  wherein said RPBLAs improve antigen delivery to antigen-presenting cells. 
     
     
         33 . The vaccine or inoculum according to  claim 27  wherein said RPBLAs improve antigen processing and presentation to antigen-presenting cells. 
     
     
         34 . The vaccine or inoculum according to  claim 27  wherein the vaccine or inoculum is for oral delivery. 
     
     
         35 . The vaccine or inoculum according to  claim 27  wherein the vaccine or inoculum is for parenteral delivery. 
     
     
         36 . An immunogenic composition comprising a recombinant protein body-like assembly (RBPLA) or a membrane-less RPBLA and a pharmaceutically acceptable diluent. 
     
     
         37 . The immunogenic composition of  claim 36 , wherein the RPBLA or the membrane-less RPBLA comprises a recombinant fusion protein, and the recombinant fusion protein comprises a protein body-inducing sequence and an adjuvant. 
     
     
         38 . The immunogenic composition of  claim 36 , wherein the RPBLA or the membrane-less RPBLA comprises a recombinant fusion protein, and the recombinant fusion protein comprises a protein body-inducing sequence- and an antigen. 
     
     
         39 . A method of making the immunogenic composition of  claim 36  comprising dissolving or dispersing an RPBLA or a membrane-less RPBLA in a pharmaceutically acceptable diluent. 
     
     
         40 . A method of inoculating an animal comprising administering the immunogenic composition of  claim 36  to the animal. 
     
     
         41 . The method of  claim 36 , further comprising administering an antigen. 
     
     
         42 . A method of stimulating a B-cell or T-cell comprising contacting the cell with the RPBLA of  claim 16 . 
     
     
         43 . A method for promoting cellular uptake comprising contacting an antigen presenting cell with the RPBLA of  claim 16 . 
     
     
         44 . The method of  claim 43 , wherein said cellular uptake occurs via phagocytosis. 
     
     
         45 . The method of  claim 43 , wherein the antigen-presenting cell is a macrophage or dendritic cell.