Part:BBa_K2607001:Design

HB-EGF/Tar Receptor (HT) Device

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 1294
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1434
  

Design Notes

Figure 1. The selected segment of Tar protein. The functional intracellular domain of Tar is shown as yellow box, blue box is transmembrane domain and orange box is periplasmic domain. Selected Tar domain expands from 1^st-33^rd amino acids and 191^st-553^rd amino acids. Modification of binding domain is located between 33^rd–191^st amino acids.

In HB-EGF, the part that serves as binding domain for diphtheria exotoxin predominantly located in the extracellular environment. Therefore, the domain, expands between 20^th–160^th amino acid, was selected from natural HB-EGF protein. On the other hand, the Tar domain that are functions to establish intracellular chemotactic signalling includes NdeI cutting-site (around 257^th amino acid) until the utmost C-terminal of the protein (the 553^rd amino acid). By those factors, our team also selected Tar domains involving the 1^st–33^rd and 191^st–553^rd amino acid as part of chimeric protein (Figure 1).

Figure 2. The graph above explains the result of HB-EGF/Tar orientation, which began from C-terminus (left) to N-terminus (right). Y-axis pictured the possibility of n^th amino acid on protein located somewhere between transmembrane (red part), intracellular (blue line), and extracellular (pink line). There is also a diagram located above the graph that represent the most possible location of each domain (with elongated box).

Figure 3. Molecular comparation of HB-EGF native protein (left) with the HB-EGF/Tar fusion (right). The pink-coloured domain is intracellularly located as the N-terminus, yellow-coloured domain for the transmembrane one. Then, purple-coloured could be a sign as the extracellular domain, finally folding into transmembrane and back to cytoplasm with orange-coloured and cyan-coloured domain respectively.

Our team have predicted the HB-EGF/Tar protein orientation in the Escherichia coli membrane. For this purpose, server TMHMM and OPM Membrane, are utilized to predict protein orientation (Figure 2 and 3). Conceptual hypothesis about the chimera protein is that it should begin its orientation of C-terminus in cytoplasm, then continued to fold into transmembrane and extracellular sites, as well as re-folding towards cytoplasm. From the results, it could be concluded that the protein was oriented as expected in the hypothesis. Therefore, the usage of chimera protein is predicted to be functional anatomically.

Figure 4. Formula for environment energy (E). E_rep and E_attr denote as repulsive and attractive contributions to the van der Waals interaction energy. Additionally, E_elec means an electrostatic energy that occur during both protein interaction. EDARS is a pairwise structure-based potential constructed by the Decoys of the Reference State (DARS) approach, and it primarily represents desolvation contributions (i.e. the free energy change due to the removal of the water molecules from the interface).

After deciding sequence combination of amino acids in model chimera HB-EGF/Tar protein, analyzing the interaction of both fusion protein and diphtheria exotoxin is extremely important to ensure functional ligand-receptor system. The basic concept of interaction modelling is that the protein will be bound to each other well if it causes the ‘environment’ energy (termed by E parameter; calculated by formula in Figure 4) being lowered down. In this part, our team sent the respective sequence to ClusPro website for further analyzing.

Source

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