Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors.
A cellular assay system for measuring the activity of cytoplasmically expressed anti-GCN4 scFv fragments directed against the Gcn4p dimerization domain was established in the budding yeast Saccharomyces cerevisiae. The inhibitory potential of different constitutively expressed anti-GCN4 scFv intrabodies was monitored by measuring the activity of beta-galactosidase expressed from a GCN4-dependent reporter gene. The in vivo performance of these scFv intrabodies in specifically decreasing reporter gene activity was related to their in vitro stability, measured by denaturant-induced equilibrium unfolding. A framework-engineered stabilized version showed significantly improved activity, while a destabilized point mutant of the anti-GCN4 wild-type showed decreased effects in vivo. These results indicate that stability engineering can result in improved performance of scFv fragments as intrabodies. Increasing the thermodynamic stability appears to be an essential factor for improving the yield of functional scFv in the reducing environment of the cytoplasm, where the conserved intradomain disulfides of antibody fragments cannot form.
Wörn
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Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Auf der Maur
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Escher
D
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Honegger
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Barberis
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Plückthun
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D000595:Amino Acid Sequence; D000908:Antibodies, Fungal / Q000276:immunology*; D003593:Cytoplasm / Q000276:immunology*; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000276:immunology*; D017930:Genes, Reporter; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011494:Protein Kinases / Q000276:immunology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
D000908:Antibodies, Fungal; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D007135:Immunoglobulin Variable Region; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases