| 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
|
| Resource |
| 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) |
