Protein_Domain

Part:BBa_K3582002

Designed by: Avadhoot Jadhav   Group: iGEM20_IISER-Pune-India   (2020-10-18)
Revision as of 20:52, 27 October 2020 by Varsha J (Talk | contribs) (→‎Characterisation)


CD36 (HUMAN PLATELET GLYCOPROTEIN 4)

CD36 is a scavenger receptor that plays a crucial role in fatty acid metabolism, innate immunity and angiogenesis. This glycoprotein molecule is popularly known for its versatile nature as it targets a broad range of ligands. Several ligands that are a potential target of CD36 include thrombospondin, fibronectin, collagen or amyloid-beta as well as of lipidic nature such as oxidized low-density lipoprotein (oxLDL), anionic phospholipids, long-chain fatty acids and bacterial diacylated lipopeptides. Their multivalent nature allows them to engage multiple receptors that play an important role in signal transduction and forming complexes. CD36 is found to be a major player in the following processes:

  • 1]Angiogenesis
  • 2]Inflammatory response
  • 3]Fatty acid metabolism
  • 4]Proximal absorption of dietary fatty acids and cholesterol
  • 5]Activation of signal pathways via MAPK1/3 (ERK1/2), TICAM1, MYD88 etc

It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. The scavenger receptor, CD36 binds to many classes of CIDRa domain (CIDRa2-6). It is also the host receptor most commonly found to interact with parasite isolates from patients and with laboratory-adapted parasite strains. Interestingly, approximately 84% of PfEMP1 proteins contain domains predicted to bind to CD36, making this the most common adhesion phenotype. The increase in the frequency of CD36 binding shows that it is advantageous to the parasite, allowing the avoidance of splenic clearance. CD36 binding may also benefit the parasite by providing a mechanism to reduce dendritic cell-mediated T-cell activation, hampering the capacity of the host immune system to clear the infection. So, in order to study these interactions between CD36 and PfEMP1 CIDR domain, we plan to synthesize the CD36 interaction protein using this biobrick and purify it using the grafted strep II tag.

Figure 1. Structure of CD36 human platelet glycoprotein visualized using Chimera (PDB:5LGD-Fu-Lien Hsieh1 et al.)

Characterisation

The EtBr gel below completes the cloning and characterisation of this biobrick.

  • The cloning of the Human Platelet Glycoprotein 4 (PDB ID: 5LGD) was achieved in 4 days after receiving the raw synthesised DNA sequences from IDT on 23rd October 2020.
Figure 1. Gel image depicting successful transformation.)

Conclusion - The band in the first column indicates the final cloned plasmid with the gene coding for the Human Platelet Glycoprotein 4 (size - 6652bp). The second column is a NEB 1kb ladder. The third column is a control with an empty plasmid - its band indicates that it is in the supercoiled state. With these results, we have successfully cloned the Human Platelet Glycoprotein 4 gene into the pET28-a(+) plasmid vector.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 169
    Illegal AgeI site found at 1129
    Illegal AgeI site found at 1255
    Illegal AgeI site found at 1303
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1418

References

  • 1] The structural basis for CD36 binding by the malaria parasite.

Fu-Lien Hsieh1, Louise Turner2, Jani Reddy Bolla3, Carol V. Robinson3, Thomas Lavstsen2 & Matthew K. Higgins1

  • 2]Racemic and Quasi-Racemic X-ray Structures of Cyclic Disulfide-Rich Peptide Drug Scaffolds**

Conan K. Wang, Gordon J. King, Susan E. Northfield, Paola G. Ojeda, and David J. Craik*



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