Improving the stability of a foreign protein in the periplasmic space of Escherichia coli.
An efficient expression/export vector comprising the entire phoS (phosphate binding protein) gene fused to a synthetic gene encoding the human growth hormone releasing factor (mhGRF) has recently been constructed [1]. The hybrid protein (PhoS-mhGRF) was exported to the periplasmic space. However, in this location proteolytic degradation occurred at the C-terminal region. Phenylmethylsulfonyl fluoride (PMSF) increased the stability of the hybrid protein indicating that a serine protease may be involved in the proteolytic cleavage. The correct export and subsequent degradation of the recombinant protein in the periplasmic space were demonstrated in situ using double immunogold labeling on ultrathin sections. Using a phoS-based expression/export vector in the presence of PMSF, 2-4 mg of hybrid protein per liter of culture could be obtained.
D015153:Blotting, Western; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D008666:Metalloendopeptidases; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; D008666:Metalloendopeptidases; C046583:pitrilysin