Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins.
Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive L-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M L-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of E. coli.
Schäffner
J
J
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany.
Winter
J
J
Rudolph
R
R
Schwarz
E
E
D003470:Culture Media; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005978:Glutathione / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D004220:Disulfides; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D011384:Proinsulin; D010959:Tissue Plasminogen Activator; D005978:Glutathione