biostudies-literatureKvam ENIAID NIH HHSNINDS NIH HHS489-98https://www.ebi.ac.uk/biostudies/studies/S-EPMC2865363biostudies-literatureUnknown23(6)Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge atProtein engineering, design & selection : PEDSPhysico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.PMC28653635R21 NS061257-025R01 NS053912-04U54 AI057159N01 AI30050Kvam EMesser AShoemaker CBSierks MRfalsePhysico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at2010-01-01T00:00:00Z2010 Jun2026-05-02T23:01:25.682Z2026-04-07T18:36:44.621ZS-EPMC28653632037869910.1093/protein/gzq022