Materials and methods involving conditional retention domains

Abstract

Materials and methods involving conditional retention domains (CRDs) are disclosed. Also disclosed are fusion proteins containing CRDs and cells expressing such fusion proteins. In addition, the invention provides novel methods for producing target proteins in vivo using fusion proteins containing conditional retention domains and methods for identifying novel CRDs.

What is claimed:  
     
         1 . A cell containing a recombinant nucleic acid encoding a fusion protein comprising at least one conditional retention domain (“CRD”) and at least one additional domain that is heterologous thereto.  
     
     
         2 . A cell of  claim 1  wherein the fusion protein contains more than one CRD.  
     
     
         3 . A cell of  claim 1  wherein the fusion protein molecules form aggregates with one another in the absence of a ligand which binds to the CRD.  
     
     
         4 . A cell of  claim 1  wherein the CRD is derived from retinol binding protein, FKBP, IgM or alpha1-antitrypsin.  
     
     
         5 . A cell of  claim 4  wherein the CRD comprises an FKBP domain with an amino acid replacement at F36 or W59.  
     
     
         6 . A cell of  claim 5  wherein the CRD comprises an FKBP domain containing the mutation F36M or W59V.  
     
     
         7 . A cell of  claim 1  wherein the heterologous domain of the fusion protein comprises the polypeptide sequence of a protein of interest.  
     
     
         8 . A cell of  claim 7  wherein the protein of interest is a hormone, an endorphin, an antibody or an immunogen.  
     
     
         9 . A cell of  claim 8  wherein the protein of interest is selected from the group consisting of insulin, parathyroid hormone and beta-endorphin.  
     
     
         10 . A cell of  claim 1  wherein the fusion protein comprises an enzymatic cleavage site.  
     
     
         11 . A cell of  claim 10  wherein the cleavage site is a furin cleavage site.  
     
     
         12 . A cell of  claim 11  wherein the furin cleavage site comprises the amino acid sequence SARNRQKR (SEQ ID NO. 1).  
     
     
         13 . A cell of  claim 1  wherein the fusion protein further comprises a secretory signal sequence.  
     
     
         14 . A cell of  claim 13  wherein the secretory signal sequence is the signal sequence from the human growth hormone gene.  
     
     
         15 . A cell of  claim 1  wherein the fusion protein comprises a secretory signal sequence, at least one conditional retention domain, a furin cleavage site, and a polypeptide sequence of interest.  
     
     
         16 . A cell of  claim 15  wherein the fusion protein comprises a secretory signal sequence from human growth hormone, three F36M FKBP domains, a human stromelysin-3 furin cleavage site, and a polypeptide sequence of interest.  
     
     
         17 . A cell of  claim 1  wherein the fusion protein comprises a lysosomal targeting signal.  
     
     
         18 . A cell of  claim 1  wherein the cells are mammalian cells.  
     
     
         19 . A cell of  claim 1  wherein the cells are of human origin.  
     
     
         20 . A cell of  claim 1  wherein the cells are primary cells.  
     
     
         21 . A cell of  claim 1  wherein the cells are in an animal.