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- W2113485885 abstract "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 VHHs in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and weak hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of principle, we demonstrate that the soluble expression of an aggregation-prone positively charged intrabody is modestly enhanced via cis or trans acidification using highly charged peptide tags (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use." @default.
- W2113485885 created "2016-06-24" @default.
- W2113485885 creator A5019220405 @default.
- W2113485885 creator A5027047896 @default.
- W2113485885 creator A5030176363 @default.
- W2113485885 creator A5077133720 @default.
- W2113485885 date "2010-04-08" @default.
- W2113485885 modified "2023-10-03" @default.
- W2113485885 title "Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm" @default.
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- W2113485885 doi "https://doi.org/10.1093/protein/gzq022" @default.
- W2113485885 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2865363" @default.
- W2113485885 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20378699" @default.
- W2113485885 hasPublicationYear "2010" @default.
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