Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.
Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96, 13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.
Levy
R
R
Institute for Cell and Molecular Biology, University of Texas, Austin, 79712, USA.
Weiss
R
R
Chen
G
G
Iverson
B L
BL
Georgiou
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D018833:Chaperonins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D003593:Cytoplasm; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004077:Digoxin / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D005980:Glutathione Reductase / Q000235:genetics*; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D007140:Immunoglobulin Fab Fragments; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; C072721:Skp protein, E coli; D004077:Digoxin; D004247:DNA; D005980:Glutathione Reductase; D018833:Chaperonins; D019704:Protein Disulfide-Isomerases