Chaperone expression genomes

Abstract

A recombinant genome comprising polynucleotides encoding at least two additional molecular chaperones and a protein of interest, recombinant baculovirus vectors providing molecular chaperones and a method for producing a foreign protein using said genomes and vectors.

1 . An isolated recombinant baculovirus vector comprising polynucleotides encoding a) at least two molecular chaperones, wherein a first chaperone is a lectin binding chaperone and a second chaperone which participates in the formation of disulfide bonds, and b) a foreign polynucleotide sequence of interest, wherein the polynucleotides are expressed from one or more cassettes within the recombinant vector, and wherein polynucleotides encoding the chaperones and the foreign polynucleotide sequence of interest are separated by at least 1000 nucleotides.  
     
     
         2 . The recombinant vector of  claim 1 , wherein a foreign polynucleotide sequence of interest allows for insertion of another polynucleotide sequence of interest and selection for vectors with the inserted polynucleotide sequence encoding for any protein of interest.  
     
     
         3 . The recombinant vector of  claim 1 , wherein a foreign polynucleotide sequence of interest encodes a reporter protein allowing for convenient negative selection of recombinant vectors expressing any protein of interest.  
     
     
         4 . The recombinant vector of  claim 1 , wherein the foreign polynucleotide sequence of interest encodes influenza virus hemagglutinin.  
     
     
         5 . The recombinant vector of  claim 1 , further comprising a polynucleotide encoding a reporter protein.  
     
     
         6 . The recombinant vector of  claim 5 , wherein the reporter protein is GFP.  
     
     
         7 . The recombinant vector of  claim 1 , wherein the first chaperone is selected from calnexin, calreticulin, ERGIC-53, ERGL, VIP36, or VIPL.  
     
     
         8 . The recombinant vector of  claim 1 , wherein a foreign polynucleotide sequence encoding a foreign protein of interest is separated from a polynucleotide sequence encoding molecular chaperones by at least: 
 a) 3,000 nucleotides; or    b) 9,000 nucleotides    
     
     
         9 . The recombinant vector of  claim 1 , wherein at least one chaperone is protein disulfide isomerase (PDI).  
     
     
         10 . The recombinant vector of  claim 1 , wherein at least one chaperone is calreticulin.  
     
     
         11 . The recombinant vector of  claim 1 , wherein the at least two chaperones are calreticulin and PDI.  
     
     
         12 . The recombinant vector of  claim 1 , having polynucleotides encoding for at least one molecular chaperone inserted into an intergenic region or non-essential gene.  
     
     
         13 . The recombinant vector of  claim 1 , wherein a site for insertion of a gene encoding a protein of interest or molecular chaperone contains a unique restriction endonuclease site comprising six or more nucleotides for specific recognition by a restriction endonuclease.  
     
     
         14 . The recombinant vector of  claim 1 , wherein the vector sequence or the genes encoding molecular chaperones are mutated in order to allow unique cleavage of the vector at the site intended to insert a protein of interest or a chaperone.  
     
     
         15 . The recombinant vector of  claim 1 , having a polynucleotide sequence of interest inserted into a polyhedrin site and a polynucleotide sequence encoding for at least one molecular chaperone inserted into a non-polyhedrin site.  
     
     
         16 . The recombinant vector of  claim 15 , wherein the non-polyhedrin site is either an intergenic region or a non-essential gene.  
     
     
         17 . The recombinant vector of  claim 15 , wherein the non-polyhedrin site is EGT, or p10 or both.  
     
     
         18 . The recombinant vector of  claim 17 , where the chaperone inserted into the p10 site is PDI.  
     
     
         19 . A method for producing a foreign protein comprising the steps of: 
 a) introducing a recombinant vector of  claim 1  into host cells;    b) culturing cells harboring a recombinant vector of  claim 1  under conditions which allow expression of the foreign protein and the molecular chaperones at levels suitable for stabilization and/or solubilization of the foreign protein; and    c) isolating the foreign protein from culture.    
     
     
         20 . A method of  claim 19 , further comprising introducing at least one second vector into appropriate host cells, 
 a) wherein the at least one second vector encodes one or more molecular chaperone selected from a lectin binding chaperone and chaperone which participates in the formation of disulfide bonds;    b) wherein the at least one second vector encodes one or more proteins of interest; and    c) wherein the at least one second vector encodes one or more molecular chaperones and one or more proteins of interest.    
     
     
         21 . A method of  claim 19 , wherein a foreign polynucleotide sequence of interest is operably linked to a polynucleotide sequence encoding insect or baculovirus signal peptide selected from the group comprising silk fibroin coding sequence, wasp  Pimpla hypochondriaca  cysteine-rich venom protein coding sequence, Baculovirus ecdysteroid UDP-glycosyltransferase (EGT) coding sequence and Honeybee melittin coding sequence.