TY - JOUR
T1 - Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm
AU - Kvam, Erik
AU - Sierks, Michael
AU - Shoemaker, Charles B.
AU - Messer, Anne
N1 - Funding Information:
This work was supported by the National Institutes of Health (5R21 NS061257-02 to A.M. and M.R.S.; 5R01 NS053912-04 to A.M. and N01 AI30050 and U54 AI057159 to C.B.S.), the Michael J. Fox Foundation to M.R.S., and the Arizona Biomedical Research Foundation to M.R.S. Summer student work on this project was supported by the NSF-REU Program (DBI0243690) to the Wadsworth Center.
PY - 2010/6
Y1 - 2010/6
N2 - 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.
AB - 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.
KW - V
KW - intrabody
KW - intracellular antibody
KW - protein aggregation
KW - scFv
UR - http://www.scopus.com/inward/record.url?scp=77955190783&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77955190783&partnerID=8YFLogxK
U2 - 10.1093/protein/gzq022
DO - 10.1093/protein/gzq022
M3 - Article
C2 - 20378699
AN - SCOPUS:77955190783
SN - 1741-0126
VL - 23
SP - 489
EP - 498
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 6
ER -