Difference between revisions of "Part:BBa K3117029:Design"
 
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===Design Notes===
 
===Design Notes===
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The sequence contains a C-terminal His-tag for easy purification and detection. Secretion of the protein is ensured by an Igk leader. When the protein passes the membrane, the leader segment is cleaved off. By connecting the variable regions of the heavy and the light chain of an anti-CD3 antibody with a short GGGGS linker (BBa_K3117004), the scFv retains its antigen-binding ability and is much smaller than a conventional antibody. Thus, it is well suited as part of a fusion protein with another effector. The SpyTag attached to the scFv belongs to the SpyTag/SpyCatcher system and is one part of the FbaB protein of Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyCatcher (BBa_K3117016), they bind covalently. This allows our part to be used in a modular manner in combination with other molecules carrying the SpyCatcher.
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[[File:G4S_4_Linker.gif|thumb|right|200px|'''Figure 1''': Modeling of the 4x[GGGS] linker]]
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The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (<partinfo>BBa_K3117020</partinfo>, <partinfo>BBa_K3117021</partinfo>, <partinfo>BBa_K3117022</partinfo>, <partinfo>BBa_K3117023</partinfo>), which are connected by a flexible [GGGGS]4 linker (<partinfo>BBa_K3117004</partinfo>). The scFvs are formed by the variable regions of the heavy and light chain of the respective antibodies that are connected by either a [GGGGS]4 or [GGGGS]3 linker (<partinfo>BBa_K3117004</partinfo>, <partinfo>BBa_K3117028</partinfo>). The sequence contains a C-terminal His-Tag (<partinfo>BBa_K3117005</partinfo>) for easy purification and detection (Schmitt et al., 1993). Secretion of the protein is ensured by an Igk leader (<partinfo>BBa_K3117006</partinfo>). When the protein passes the membrane, this leader domain is cleaved off.
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===Source===
 
===Source===
  
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===References===
  
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===References===
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1. Schmitt, J., Hess, H., & Stunnenberg, H. G. (1993). Affinity purification of histidine-tagged proteins. Molecular biology reports, 18(3), 223-230.

Latest revision as of 22:24, 21 October 2019


scFv bispecific antibody against GPA33 and CD3 codon optimized for CHOs


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 947
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Figure 1: Modeling of the 4x[GGGS] linker

The part consists of humanized anti-CD3 and anti-GPA33 single-chain variable fragments (scFv) (BBa_K3117020, BBa_K3117021, BBa_K3117022, BBa_K3117023), which are connected by a flexible [GGGGS]4 linker (BBa_K3117004). The scFvs are formed by the variable regions of the heavy and light chain of the respective antibodies that are connected by either a [GGGGS]4 or [GGGGS]3 linker (BBa_K3117004, BBa_K3117028). The sequence contains a C-terminal His-Tag (BBa_K3117005) for easy purification and detection (Schmitt et al., 1993). Secretion of the protein is ensured by an Igk leader (BBa_K3117006). When the protein passes the membrane, this leader domain is cleaved off.

Source

References

1. Schmitt, J., Hess, H., & Stunnenberg, H. G. (1993). Affinity purification of histidine-tagged proteins. Molecular biology reports, 18(3), 223-230.