Engineered turns of a recombinant antibody improve its in vivo folding.
Using recombinant antibodies functionally expressed by secretion to the periplasm in Escherichia coli as a model system, we identified mutations located in turns of the protein which reduce the formation of aggregates during in vivo folding or which influence cell stability during expression. Unexpectedly, the two effects are based on different mutations and could be separated, but both mutations act synergistically in vivo. Neither mutation increases the thermodynamic stability in vitro. However, the in vivo folding mutation correlates with the yield of oxidative folding in vitro, which is limited by the side reaction of aggregation. The in vivo folding data also correlate with the rate and activation entropy of thermally induced aggregation. This analysis shows that it is possible to engineer improved frameworks for semi-synthetic antibody libraries which may be important in maintaining library diversity. Moreover, limitations in recombinant protein expression can be overcome by single amino acid substitutions.
Knappik
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Biochemisches Institut, Universität Zürich, Switzerland.
Plückthun
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D015153:Blotting, Western; D004926:Escherichia coli / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010767:Phosphorylcholine / Q000276:immunology; D017354:Point Mutation; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D010767:Phosphorylcholine; D014508:Urea; D007535:Isomerases; D019704:Protein Disulfide-Isomerases