Difference between revisions of "Part:BBa K5271011"
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::https://static.igem.wiki/teams/5271/part-figures/her2-fc-egfr-structure1.png
 
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::'''The AlphaFold predicted structure of HER2-Fc-EGFR.''' Red: His-tag; Green: HER2 nanobody; Grey: linker; Blue: Fc protein; Orange: EGFR nanobody.
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::'''Figure 1. The AlphaFold predicted structure of HER2-Fc-EGFR.''' Red: His-tag; Green: HER2 nanobody; Grey: linker; Blue: Fc protein; Orange: EGFR nanobody.
  
  

Revision as of 09:54, 30 September 2024


HER2nb-Fc-EGFRnb

A nanobody composed of two binding regions of EGFR and HER2 linked by a fragment of Fc region of the heavy chain of human IgG.


Profile

  • Name: HER2nb-Fc-EGFRnb
  • Base Pairs: 1515 bp
  • Amino acid: 527 a.a
  • Origin: Synthetic
  • Property: A dual targeting nanobody with a molecular weight of 55 kDa consisting of a Fc fragment and two HER2 and EGFR nanobody binding domains that specifically bind to EGFR and HER2.



Usage and Biology

her2-fc-egfr-structure1.png
Figure 1. The AlphaFold predicted structure of HER2-Fc-EGFR. Red: His-tag; Green: HER2 nanobody; Grey: linker; Blue: Fc protein; Orange: EGFR nanobody.




Expression

sds-gel-her2-fc-egfr-1.png
Figure 2. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total 30℃ HER2-FcC-EGFR in 6hr uninduced fraction; lane 3, Total 30℃ HER2-FcC-EGFR in 6hr 0.5mM IPTG induced fraction; lane 4, Total 30℃ HER2-FcC-EGFR overnight uninduced fraction; lane 5, Total 30℃ HER2-FcC-EGFR overnight 0.5mM IPTG induced fraction; lane 6, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 7, Total 16℃ HER2-FcC-EGFR overnight uninduced fraction; lane 8, Total 16℃ HER2-FcC-EGFR overnight 0.5mM IPTG induced fraction. ON = overnight.​



sds-gel-her2-fc-egfr-2.png
Figure 3. Protein expression of HER2-FC-EGFRnb. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total 25℃ HER2-FC-EGFR-1 in 6hr uninduced fraction; lane 3, Total 25℃ HER2-FC-EGFR-1 in 6hr 0.5mM IPTG induced fraction; lane 4, Total 25℃ HER2-FC-EGFR-1 overnight uninduced fraction; lane 5, Total 25℃ HER2-FC-EGFR-1 overnight 0.5mM IPTG induced fraction; lane 6, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 7, Total 25℃ HER2-FcC-EGFR-2 in 6hr uninduced fraction; lane 8, Total 25℃ HER2-FcC-EGFR-2 in 6hr 0.5mM IPTG induced fraction; lane 9, Total 25℃ HER2-FcC-EGFR-2 overnight uninduced fraction; lane 10, Total 25℃ HER2-FcC-EGFR-2 overnight 0.5mM IPTG induced fraction. ON = overnight, hr= Hours


sds-gel-her2-fc-egfr-3.png
Figure 4. Protein expression of HER2-FcC-EGFRnb. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Soluble 25℃ HER2-FcC-EGFR-1 in 6hr 0.5mM IPTG induced soluble fraction; lane 3, Insoluble 25℃ HER2-FcC-EGFR-1 in 6hr IPTG induced insoluble fraction; lane 4, Soluble 25℃ HER2-FcC-EGFR-1 overnight IPTG induced soluble fraction; lane 5, Insoluble 25℃ HER2-FcC-EGFR-1 overnight IPTG induced insoluble fraction; lane 6, Soluble 25℃ HER2-FcC-EGFR-2 in 6hr IPTG induced soluble fraction; lane 7, Insoluble 25℃ HER2-FcC-EGFR-2 in 6hr IPTG induced insoluble fraction; lane 8, Soluble 25℃ HER2-FcC-EGFR-2 overnight IPTG induced soluble fraction; lane 9, Insoluble 25℃ HER2-FcC-EGFR-2 overnight IPTG induced insoluble fraction; lane 10, Soluble 25℃ HER2-FcC-EGFR-2 in 6hr uninduced soluble fraction. ON = overnight; Sol = soluble; Insol = insoluble​



sds-gel-her2-fc-egfr-4.png
Figure 5. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Total HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 3, Total HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 4, Total HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 5, Total HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 6, Total HER2-FcC-EGFR in 25℃ with 0.2mM IPTG induced fraction; lane 7, Total HER2-FcC-EGFR in 25℃ uninduced fraction; lane 8, Insoluble HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 9, Insoluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 10, Insoluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction. Insol = insoluble​




sds-gel-her2-fc-egfr-5.png
Figure 6. Protein expression of HER2-FcC-EGFR. Lane 1, Biorad Precision Plus Protein™™ Unstained Protein Standards; lane 2, Soluble HER2-FcC-EGFR in 16℃ with 0.2mM IPTG induced fraction; lane 3, Soluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 4, Soluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 5, Soluble HER2-FcC-EGFR in 16℃ with uninduced fraction; lane 6, Soluble HER2-FcC-EGFR in 25℃ with 0.2mM IPTG induced fraction; lane 7, Soluble HER2-FcC-EGFR in 25℃ uninduced fraction; lane 8, Insoluble HER2-FcC-EGFR in 16℃ with 0.2mM uninduced fraction; lane 9, Insoluble HER2-FcC-EGFR in 16℃ with 0.5mM IPTG induced fraction; lane 10, Insoluble HER2-FcC-EGFR e in 25℃ with 0.2mM uninduced fraction. Sol=soluble; Insol = insoluble







Reference

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 154
    Illegal NheI site found at 1615
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 643
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 820
    Illegal AgeI site found at 1275
    Illegal AgeI site found at 1407
  • 1000
    COMPATIBLE WITH RFC[1000]