Methods and Compositions for Protein Expression and Purification

Abstract

Methods for enhancing expression levels and secretion of heterologous fusion proteins in a host cell are disclosed.

1 . A kit comprising a recombinant vector containing a nucleic acid sequence encoding a UBL molecule selected from the group of SUMO, RUB, HUB, URM1, and ISG15 operably linked to a promoter suitable for expression in the desired host cell and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest in-frame with the nucleic acid sequence encoding the UBL molecule.  
     
     
         2 . The kit of  claim 1 , wherein said kit further comprises host cells suitable for expression of said vector.  
     
     
         3 . The kit of  claim 2 , wherein said host cells are selected from the group of yeast cells,  E. coli,  insect cells, and mammalian cells.  
     
     
         4 . The kit of  claim 1 , wherein said kit further comprises a kit which comprises reagents for altering the nucleic acid encoding said protein of interest to generate amino termini which are different from those native to the wild-type protein.  
     
     
         5 . The kit of  claim 4 , wherein said kit comprises reagents suitable for site-directed mutagenesis.  
     
     
         6 . The kit of  claim 5 , wherein said kit comprises oligonucleotides for performing oligonucleotide-based site-directed mutagenesis.  
     
     
         7 . A kit for purification of a protein from a host cell comprising: 
 i) a recombinant vector containing a nucleic acid sequence encoding a UBL molecule selected from the group of SUMO, RUB, HUB, URM1, and ISG15 operably linked to a promoter suitable for expression in the desired host cell, a nucleic acid sequence encoding for a purification tag in-frame with the nucleic acid sequence encoding the UBL molecule, and a multiple cloning site suitable for cloning a nucleic acid encoding the protein of interest in-frame with the nucleic acid sequence encoding the UBL molecule, and    ii) a protease composition capable of cleaving the UBL molecule from the fusion protein.    
     
     
         8 . The kit of  claim 7 , wherein said kit further comprises host cells suitable for expression of said vector.  
     
     
         9 . The kit of  claim 8  wherein said host cell is selected from the group of yeast cells,  E. coli,  insect cells, and mammalian cells.  
     
     
         10 . The kit of  claim 7  further comprising: 
 i) a solid support for binding the purification tag,    ii) lysis buffers,    iii) wash buffers,    iv) elution buffers,    v) cleavage buffers, and    vi) instruction material.