Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting
- Part Collection
Single chain variable fragment (scFV) recognizing Ras - Part:BBa_K3573000
ScFv(Ras) is a single chain variable fragment that is designed to recognize human Ras protein. ScFv(Ras) was engineered from scFv(F8) that is hyperstable and is functional in the reducing environment inside the cell.
This year's project is a continuation of 2019's project. Last year, our team designed a cell-penetrating hyperstable single chain variable fragment (scFv) by attaching cell-penetrating peptide (CPP) to the N-terminus of scFv(P5). CPP-scFv(P5) is derived from scFv(F8) that is inherently hyperstable. We have shown that CPP-scFv(P5) can enter the cell and recognize its cognate target protein, lysozyme in a reducing environment.
We have also designed a hyperstable, cell-penetrating scFv targeting Ras protein (scFv(Ras)) based on the structure of Ras - anti-Ras antibody (PDB ID: 2vh5). This year we have cloned, expressed, purified and performed a binding assay of designed scFv(Ras) and HRas, using real proteins.
2vh5 structure showing HRas: antibody interface residues
2VH5 Heavy + Light Chains
EVQLLESGGGLVQPGGSLRLSAAASGFTFSTFSMNWVRQAPGKGLEWVSYISRTSKTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYVARGRFFDY WGQGTLVTVSIQMTQSPSSLSASVGDRVTITVRASQSISSYLNWYQQKPGEAPKLLIYSASVLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYAQQSVMIP MTFGQGTKVE
scFv(F8) with CPP and Linker
scFv(F8) with CPP and Linker, 2VH5's antigen binding residues grafted
QVQLQESGGDLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINRTGKTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGRFFDY WGQGTTVTVSS GGGGSGGGGSGGGGS
This DNA was synthesized
Single chain variable fragment with CPP recognizing Ras - Part:BBa_K3573001
This part is a Single chain variable fragment that recognizes Ras by having a cell-penetrating peptide (CPP) attached at the N-terminus.
Last year, our team modeled the structure of CPP-scFv(Ras) using the program Modeller. Modeller searched the database and used similar structures (PDB ID: 5B3N, 6NJL, 5GS3, 6G8R and 3UYP) to build a homology model. Based on the results of the homology modelling, we have decided to synthesize the gene for CPP-scFv(Ras) for protein production and binding assays. CPP (BBa_K3090000) was attached to scFv(Ras)(BBa_3573000) for entry into the cell.
Human Ras G12V mutant - Part:BBa_K3573002
This part is a Human Ras protein with G12V mutation. Ras is a well known oncogene involved in cell cycle control.
Ras signaling pathways. Ras signaling is involved in numerous cellular functions, including cell proliferation, apoptosis, migration, fate specification, and differentiation. A key Ras effector pathway is the mitogen-activated protein kinase (MAPK), Raf-MEK- ERK pathway.
EGF binds to the extracellular domain of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK). The signal is transmitted through the transmembrane domain resulting in EGFR dimerization and activation. Activated EGFR recruits the son of sevenless (SOS), a guanine nucleotide exchange factor (GEF), to its phosphorylated C-terminal tail via the adaptor proteins, SH2- adaptor protein (SHC) and growth factor receptor-bound protein 2 (Grb2).
GEF exchanges GDP by GTP, activating Ras. Active, GTP- loaded Ras dimerizes and binds Raf, thereby promoting Raf dimerization and activation. Active Raf dimer phosphorylates and activates mitogen-activated protein kinase kinase 1 and 2 (MEK1/2), which induces ERK1/2 activation. Transcription factor Elk-1 is among ERK1/2 many downstream phosphorylation targets. Elk-1 binds to its cofactor, a dimer of serum response factor (SRF), leading to transcription activation and cell proliferation. Active GTP-bound Ras regulates a number of signaling pathways; among these is phosphatidylinositol 3-kinase (PI3K). PI3K is a heterodimer with a regulatory (p85) and catalytic (p110) subunits (not shown here). RTKs recruit the p85 subunit of PI3K.
Ras activates p110 independently of p85 . PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidylinositol-3,4,5-trisphosphate (PIP3), a process which can be reversed by the action of phosphatase and tensin homologue (PTEN). PIP3 recruits Phosphoinositide-dependent kinase-1 (PDK1) that phosphorylates a serine/threonine kinase, Akt (also known as PKB, protein kinase B) in the plasma membrane.
This further induces the activation of the mammalian target of rapamycin (mTOR) complex, one of the major pathways leading to cell growth. This pathway plays important roles in Ras-mediated cell survival and proliferation.
Purchased from addgene
<reference> Nussinov R, Jang H, Tsai CJ. The structural basis for cancer treatment decisions. Oncotarget. 2014 Sep 15;5(17):7285-302. Review.