Part:BBa_K4960037

pPVC promoter->pvc1-pvc12->pvc13_NTD-3*GGGSG-CKGGRAKDC-3*GGGSG -pvc13_CTD->pvc14-pvc16

A flexible linker which can influence the structure of tail fiber connected to the CKGGRAKDC.

Usage and Biology

The system is a syringe-like macromolecular complex that inject protein payloads into fat cells by driving a spike through the cellular membrane. Different payloads can be load into the lumen of the inner tube behind the spike, form fusion proteins with the tube, or associate with the spike itself. Studies suggest that the tail fibre is probably involved in target recognition. [1]Our project has enabled the system to target adipocytes by modifying the tail fibre to bind to CKGGRAKDC. CKGGRAKDC is a peptide motif that homes to white fat vasculature. The studies show that CKGGRAKDC targets the white adipose vasculature without a detectable preference for any particular anatomical white fat depot. Meanwhile, the motif is preferentially internalized by a receptor in the adipose vasculature that may serve for targeted delivery of therapeutic compounds to fat.[2] After specific recognition and binding to adipocytes, it can cross the via sheath contraction and inject the desired payloads. The linker 3*GGGSG on both sides of CKGGRAKDC can affect the function and efficiency of bond and recognition by affecting the structure of the tail protein.(Fig.1)

Figure 1.AlphaFold2 based prediction of engineered PVC tail fiber trimer structure.
Structure of adipose-targeting CKGGRAKDC peptide-presenting PVC tail fiber with indicated linkers were shown.

Special Design

Use the CKGGRAKDC to target fat cells. The different linker(1*GGGSG or 3*GGGSG) on both sides of the CKGGRAKDC can affect the structure and function of the tail protein. By altering the linker on both sides of the CKGGRAKDC, we explored the most suitable structure of the tail protein for recognition and bond. At the same time, because of the existence of linker, we can use Golden Gate to replace the sequences in the middle of linker more efficiently than enzyme cutting and enzyme linking.

Sequence and Features

Tube:pvc1, pvc5, pvc7
Sheath:pvc2, pvc3, pvc4
Sheath terminator:pvc15
Tail fbres:pvc13_NTD-3*GGGSG-CKGGRAKDC-3*GGGSG -pvc13_CTD
Baseplate:pvc9, pvc11, pvc12
Spike:pvc8, pvc10
(Fig.2) Assembly method:3A Assembly

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7406
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2407
  Illegal BglII site found at 11264
  Illegal BglII site found at 14099
  Illegal BglII site found at 17020
  Illegal XhoI site found at 2691
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 11792
  Illegal NgoMIV site found at 17551
  Illegal NgoMIV site found at 17690
  Illegal AgeI site found at 6704
  Illegal AgeI site found at 9641
  Illegal AgeI site found at 10327
  Illegal AgeI site found at 10468
  Illegal AgeI site found at 11576
  Illegal AgeI site found at 17989
  Illegal AgeI site found at 19677
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1]Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7. Epub 2023 Mar 29. PMID: 36991127; PMCID: PMC10097599.
[2]Kolonin MG, Saha PK, Chan L, Pasqualini R, Arap W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med. 2004 Jun;10(6):625-32. doi: 10.1038/nm1048. Epub 2004 May 9. PMID: 15133506.