Part:BBa_K3573000:Design

Single chain variable fragment (scFV) recognizing RAS

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This year's project is a continuation of 2019's project. Last year our team (Korea_HS) designed a cell-penetrating hyperstable single chain variable fragment (scFv) by attaching cell-penetratng 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 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:

MTYTRRRFRRRRHRPRS QVQLQESGGDLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINSNGGSTFYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRRNYPY YYGSRGTFDYWGQGTTVTVSS GGGGSGGGGSGGGGS DIELTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRALNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGG GTKLEIKR ​

scFv(F8) with CPP and Linker, 2VH5's antigen binding residues grafted:

MTYTRRRFRRRRHRPRS QVQLQESGGDLVQPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLELVATINRTGKTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARGRFFDY WGQGTTVTVSS GGGGSGGGGSGGGGS DIELTQSPASLAVSLGQRATISCRASESVDSYMHWYQQKPGQPPKLLIYRALNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSVEDPWTFGGGTKL EIKR

Source

This DNA was synthesized