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

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===Design Notes===
 
===Design Notes===
  
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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.
+
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.
  
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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 designed scFv(RAS) and HRas using real proteins.
  
 
[[File:T--Korea_HS--Interface.png|500px]]
 
[[File:T--Korea_HS--Interface.png|500px]]
 
2vh5 structure showing HRas: antibody interface residues
 
2vh5 structure showing HRas: antibody interface residues
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 +
  
 
2VH5 Heavy + Light Chains:
 
2VH5 Heavy + Light Chains:

Latest revision as of 00:09, 26 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 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.

T--Korea HS--Interface.png 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

References