Difference between revisions of "Team:CCU Taiwan/Poster"

Linear array epitope (LAE) is a technique to produce tandem-repeated sequences (TRSs). After the procedure, the TRSs would be expressed as a long-chain peptide which can be cleaved by an enzyme, resulting in many identical peptides.

The procedure of LAE:

1. Design DNA oligo primers.
2. Template-repeated PCR (TR-PCR).
3. Adaptor PCR (AD-PCR).

For the primer design, TR-PCR primers need to be partially complementary to each other and AD-PCR primers need to have restriction sites.

In TR-PCR, those primers act as both primer and template and they are partially complementary. The TRSs can be produced during the PCR cycling process. In AD-PCR, restriction sites need be introduced into the products of TR-PCR by adding the AD-PCR primers in PCR.

The BioBrick of TRSs is composed of T7 promoter, lac operator, thrombin site, TRSs, and His tag.

The BioBrick of E protein is composed of a T7 promoter, lac operator, thrombin site, E protein gene, HA tag, and His tag.

The BioBrick of CLEC5A is composed of a T7 promoter, lac operator, thrombin site, CLEC5A gene, Myc tag, and His tag.

Our peptides and proteins were expressed in E. Coli with T7 promotor and lac operator, which can be induced by IPTG. The thrombin site was included to remove unnecessary sections. The His tag was used in Western blot to confirm the expression of protein, and the HA tag and myc tag were utilized for purification.

Purpose: Our goal is to design a peptide of tandem-repeated sequence (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.

Method and Results:

1. The structure of E protein (Figure 1) from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).
2. The structures of PTRSs were predicted purely based on their sequences using the ab initio method in Rosetta, and the representative structures are shown in Figure 2.
3. The most probable structure of the PTRS were identified by clustering.
4. The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown in Figure 3.

Figure 1. The homologous structure of PL046 E protein (cyan) based on the deposited structure (PDB: 1OAN) magenta.

Figure 2. The representative structures of a PTRS.

Figure 3. The best 100 results (based on the Rosetta score) of PTRS docking to PL046 E protein (red). The space above the plane (grey) indicates the external surface of the virion, where the interactions occur.

Modeling: Interaction between gold nanoparticles Purpose: A potential weakness of our design is that peptides on the gold nanoparticles and on the glass fiber compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by gold nanoparticles, there would be no sites available to interact with the peptides attached to the glass fiber. Hence, we need to understand if this situation would hinder development of our kit.

Method: We used DLVO theory to calculate the repulsion between gold nanoparticles to estimate the number of gold nanoparticles that would bind to a virus particle. The structure of dengue virus is icosahedral, and there are nine E proteins on each surface. The distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R).
DLVO theory can be described as follows, W_total(D) = W_a(D) + W_r(D) = -AR/12D + 2πεε₀RΨ_δ²exp(-κD) W_total(D): total energy
W_a(D): van der Waals interaction energy
W_r(D): electrostatic interaction energy
A: Hamaker constant = 2.5×10^-19 J
R: radius = 1.3x10^-8 m (based on the size of gold nanoparticles)
C: center to center distance
D: C-R
ε: permittivity of vacuum = 8.854x10^-12 F/m
ε₀: dielectric of solution = 80.4 (at 20°C)
Ψ_δ²: Stern layer potential (zeta potential) = 1.794 mV
κ^-1: diffuse layer thickness = 7.95x10^-10 m

We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the gold nanoparticles based on DLVO theory.

Results: The total energies are all positive, indicating repulsive forces, (Table 1) and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.

Table 1. Energies of the gold nanoparticles between representative positions.

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