The effect of folding catalysts on the in vivo folding process of different antibody fragments expressed in Escherichia coli.
The Fv and Fab fragment and both orientations of the single-chain Fv fragment (VH-linker-VL and VL-linker-VH) of an antibody can be expressed in functional form in the periplasm of Escherichia coli, but the yield of these correctly assembled proteins is limited by the periplasmic folding process. While the periplasmic E. coli disulfide isomerase DsbA is required for this assembly, its functional over-expression does not significantly change the folding limit. Similarly, the functionally over-expressed E. coli proline cis-trans isomerase does not change the amount of all but one of the antibody fragments, not even if DsbA is over-expressed as well. Therefore, aggregation steps in the periplasm appear to compete with periplasmic folding, and they may occur before disulfide formation and/or proline cis-trans isomerization takes place and be independent of their extent.
Knappik
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Max-Planck-Institut für Biochemie, Protein Engineering Group, Martinsried, Fed. Rep. Germany.
Krebber
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Plückthun
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics; D007535:Isomerases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases