Difference between revisions of "Part:BBa K3573000:Design"

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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 scFv(RAS) and HRas using real proteins.
 
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 scFv(RAS) and HRas using real proteins.
  
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[[File:T--Korea_HS--Interface.png|500px|right|2vh5 structure]]
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[[File:T--Korea_HS--Interface.png|500px|thumb|right|2vh5 structure]]
  
 
2VH5 Heavy + Light Chains:
 
2VH5 Heavy + Light Chains:

Revision as of 23:47, 25 October 2020


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 CPP-scFv(P5) by engineering 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 scFv(RAS) and HRas using real proteins.

2vh5 structure

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

References