| Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm. | |
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MedLine Citation:
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PMID: 20378699 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Erik Kvam; Michael R Sierks; Charles B Shoemaker; Anne Messer |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S. Date: 2010-04-08 |
Journal Detail:
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Title: Protein engineering, design & selection : PEDS Volume: 23 ISSN: 1741-0134 ISO Abbreviation: Protein Eng. Des. Sel. Publication Date: 2010 Jun |
Date Detail:
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Created Date: 2010-05-07 Completed Date: 2010-08-03 Revised Date: 2011-07-28 |
Medline Journal Info:
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Nlm Unique ID: 101186484 Medline TA: Protein Eng Des Sel Country: England |
Other Details:
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Languages: eng Pagination: 489-98 Citation Subset: IM |
Affiliation:
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New York State Department of Health, Wadsworth Center/ David Axelrod Institute, 120 New Scotland Ave., PO Box 22002, Albany, NY 12201-2002, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Analysis of Variance Animals Camelids, New World Cell Line Cytoplasm / metabolism* Databases, Protein Green Fluorescent Proteins Humans Hydrogen-Ion Concentration Hydrophobic and Hydrophilic Interactions Microscopy, Fluorescence Molecular Sequence Data Peptides Protein Multimerization Rats Recombinant Fusion Proteins / chemistry, genetics, metabolism Reproducibility of Results Single-Chain Antibodies / biosynthesis*, chemistry*, genetics Solubility |
| Grant Support | |
ID/Acronym/Agency:
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5R01 NS053912-04/NS/NINDS NIH HHS; 5R21 NS061257-02/NS/NINDS NIH HHS; N01 AI30050/AI/NIAID NIH HHS; U54 AI057159/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Peptides; 0/Recombinant Fusion Proteins; 0/Single-Chain Antibodies; 147336-22-9/Green Fluorescent Proteins; 98849-88-8/FLAG peptide |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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