Abstract
Targeting cytoplasmic protein–protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras—long recognised as an “undruggable” oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.
Highlights
Targeting cytoplasmic protein–protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates
Fusion of T7, enhanced green fluorescent protein (EGFP), and histidine tags to scFv-A36 or scFv-M4 increased the net negative charge of both proteins at pH 7.4 (Fig. 1a, b); the negative charge was markedly reduced at pH 7.03 (Fig. 1b) and 6.6 (Fig. 1b)
We tested the generalisability of the effects of these peptide tags on the net charge by using 94 other scFv proteins obtained from an NCBI BLAST search of the scFv-A36 sequence; we found that their effects were similar to those of scFv-A36 and scFv-M4 (Supplementary Fig. 2)
Summary
Targeting cytoplasmic protein–protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments. Screening of stable scFvs from hybridoma clones and/or introducing structural analysis-based amino acid substitutions is necessary to improve their folding and stability in the cytoplasm[8]. Researchers have investigated intrabody formats—camelids-derived single-domain antibodies (VHHs), and antibody-like fibronectin-derived proteins (FingRs) that fold stably with no disulfide bonds[9,10]. VHHs are not always stably expressed in the cytoplasm of cultured mammalian cells[4,11]; only 10−20% of cytoplasmic FingRs among clones screened from a FingR library were stable and functional[10]
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