title: Heat-shock protein fusion vectors for improved expression of soluble recombinant proteins in Escherichia coli.
abstract: Molecular chaperones or heat-shock proteins (HSPs) are protein machines that interact with unfolded or partially folded polypeptides and assist them in attaining their proper conformation. The folding reaction relies on a complex array of scaffolding effects and ATP-driven conformational changes that mediate the temporary unfolding and subsequent refolding of protein substrates. DnaK and GroEL are the two major Escherichia coli chaperones. They belong to the HSP70 and HSP60 families of proteins, respectively, and play a major role in protein folding. Here, we describe a set of bacterial expression vectors that permits the fusion of a protein of interest to DnaK or GroEL and its subsequent quantitative expression in a soluble, easily purifiable form. We also provide a set of compatible co-chaperone expression constructs that permit the simultaneous co-expression of the DnaK and GroEL physiological partners to further increase protein solubility. The system was successfully tested using the murine prion protein (PrP). Although PrP is normally insoluble when expressed in E. coli, we show that utilizing our vectors it can be produced in a soluble form as a DnaK or GroEL fusion. This system is useful for the production of a large array of proteins that fail to fold properly when expressed in E. coli.
authors.affiliation: Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA.
authors.forename: Christos A
authors.initials: CA
authors.lastname: Kyratsous
authors.forename: Christos A
authors.initials: CA
authors.lastname: Panagiotidis
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011328:Prions / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011328:Prions; D011994:Recombinant Proteins; C063842:dnaK protein, E coli