RRC ID 70015
Author Kabayama H, Takeuchi M, Tokushige N, Muramatsu SI, Kabayama M, Fukuda M, Yamada Y, Mikoshiba K.
Title An ultra-stable cytoplasmic antibody engineered for in vivo applications.
Journal Nat Commun
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.
Volume 11(1)
Pages 336
Published 2020-1-17
DOI 10.1038/s41467-019-13654-9
PII 10.1038/s41467-019-13654-9
PMID 31953402
PMC PMC6969036
MeSH Amino Acid Sequence Amino Acid Substitution Animals Antibodies / genetics Antibodies / metabolism* Behavior, Animal COS Cells Cell Proliferation Chlorocebus aethiops Cytoplasm / genetics Cytoplasm / metabolism* HEK293 Cells HeLa Cells Humans Hydrogen-Ion Concentration Male Mice Neoplasms / therapy Protein Engineering / methods* Xenograft Model Antitumor Assays
IF 12.121
DNA material pGEM-scFv-T7-A36 (RDB19420) pGEM-scFv-GFPA36 (RDB19421) pET-scFv-GFPA36 (RDB19422) pIRES-scFv-GFPA36 (RDB19423) pGEM-scFv-A36 mutant (M4) (RDB19424) pGEM-scFv-GFPA36 mutant (M4) (RDB19425) pGEM-s3Flag-scFv-A36 (RDB19426) pGEM-s3Flag-scFv-M4 (RDB19427) ER-scFv-A36-expressing vector (RDB19428)
Human and Animal Cells 293T(RCB2202) HeLa.S3(RCB0191) MIA Paca2(RCB02094)