Secretion and in vivo folding of the Fab fragment of the antibody McPC603 in Escherichia coli: influence of disulphides and cis-prolines.
Using the well-characterized antibody McPC603 as a model, we had found that the Fv fragment can be isolated from Escherichia coli as a functional protein in good yields, whereas the amount of the correctly folded Fab fragment of the same antibody produced under identical conditions is significantly lower. In this paper, we analyse the reasons for this difference. We found that a variety of signal sequences function in the secretion of the isolated chains of the Fab fragment or in the co-secretion of both chains in E.coli. The low yield of functional Fab fragment is not caused by inefficient expression or secretion in E.coli, but by inefficient folding and/or assembly in the periplasm. We compared the folding yields for the Fv and the Fab fragment in the periplasm under various conditions. Several diagnostic framework variants were constructed and their folding yields measured. The results show that substitutions affecting cis-proline residues and those affecting various disulphide bonds in the protein are by themselves insufficient to dramatically change the partitioning of the folding pathway to the native structure, and the cause must lie in a facile aggregation of folding intermediates common to all structural variants. However, all structural variants could be obtained in native form, demonstrating the general utility of the secretory expression strategy.
Skerra
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Genzentrum der Universität München, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Plückthun
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism; D004220:Disulfides / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007070:Immunoglobulin A / Q000235:genetics* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism; D007134:Immunoglobulin Switch Region / Q000235:genetics; D007536:Isomerism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004220:Disulfides; D007070:Immunoglobulin A; D007140:Immunoglobulin Fab Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011392:Proline