title: Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.
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 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 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.
authors.affiliation: New York State Department of Health, Wadsworth Center/ David Axelrod Institute, 120 New Scotland Ave., PO Box 22002, Albany, NY 12201-2002, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Kvam
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sierks
authors.forename: Charles B
authors.initials: CB
authors.lastname: Shoemaker
authors.forename: Anne
authors.initials: A
authors.lastname: Messer
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D002161:Camelids, New World; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D030562:Databases, Protein; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides; D055503:Protein Multimerization; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
chemical_list: D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D049452:Green Fluorescent Proteins; C085706:FLAG peptide