Team:Duke/Results

DESIGN STATEMENT


To design and express a significantly more thermostable Griffithsin dimer and monomer for future implementation in a diagnostic assay.

MODELING of GRIFFITHSIN


Our design began with taking the originally published sequence of griffithsin and modeling thermostable changes in the sequence. Click here for more information on our modeling.

Developing the monomer form of Grffithsin is important because it can create tandemers that are higher binding to HIV, which is important for lateral flow assay design.

DESIGNING A HIGH-EXPRESSING CONSTRUCTS


After creating an optimal amino acids sequence from the Yasara modeling, we codon optimized the sequence in E. coli via IDT. To express this protein maximum level, we placed the gene within a T7 promoter system—an adapted viral promoter that maximizes expression. This gBlock was subsequently cloned into our the LC-pSmart Kan vector, using Gibson Assembly.

IPTG INDUCED EXPRESSION


To mimic industrial expression protocols, we created a protocol to first maximize the biomass of the Griffithsin-expressing cells. Then the IPTG was added to turn on the promoter and hijack the cell’s metabolism to producing GRFT. The GRFT was then removed from the cells.

To express griffithsin (GRFT) at maximum levels, dynamic metabolic control was exercised using IPTG induction as the metabolic valve.

  1. Biomass is grown in of SM10++(max 10g/L) media for 18 hours
  2. IPTG rich SM0++ media is then added to the culture which induces:
    1. The vast majority of its metabolism shifts to protein creation as the strong promoter is turned on.
    2. Cell growth will dramatically decrease
  3. Homgenization
    1. Since Griffithsin is an intracellular protein, homogenization was chosen as the method taken to lyse the cells.
    2. Homogenization feeds a sample through small aperture where the cell is lysed by shearing

Figure: Homogenization

CONFIRMATION OF GRIFFITHSIN EXPRESSION


We ran an SDS page gel on the lysate to confirm expression of griffithsin. The protocol we used for these is in the experiments tag but it is also diagramed below.

The following gel shows the presence of griffithsin.

Figure 1: 12% Bis-Tris SDS-PAGE. Lanes: NEB 10-200 kDa Ladder Ladder (L), Empty pSmart Vector (EV), Thermostable Griffithsin Monomer (TSm), Thermostable Griffithsin Dimer (TS)

Hydropathicity Considerations


Previous studies in our lab produced the following SDS-PAGE gel confirming that Wildtype-Griffithsin runs at 12 kDa.

Figure 2: From a previous experiment.


However, our SDS-Page gels produced a band at ~35 kDa. We hypothesize that the higher band results from the increased hydrophobicity from the thermostability modifications. The hydrophobicity significantly impacts the electrophoresis of proteins because the more hydrophobic the protein is, the more SDS binds to it (http://www.pnas.org/content/106/6/1760.full). Since SDS is negatively charged, additional binding would significantly reduce the electromagnetic driving force of GRFT, causing it to run higher.

Figure 3: Thermostable Hydrophobicity scores from Expasy

Figure 4: Wild Type Hydrophobicity scores from Expasy

Using the protein modeling software,ExPASy, we made hydropathicity plots from the amino acids sequence for the wild type and the thermostable version. The area beneath the curves is directly related to the average hydrophobicity of each protein: the Protein GRAVY (grand average of hydropathy) value to quantify and compare the two versions. Positive values are associated with higher relative hydrophobicity.

Comparing the area of the plots, the thermostable variant of GRFT is much more hydrophobic than the wildtype. The wildtype had a protein GRAVY value of -0.240 while the thermostable variant had a value of -0.149. GRAVY values that are more positive are more hydrophobic. The thermostable variant is 1.6 times more hydrophobic than the wild type variant.

THERMOSTABILITY HEAT TRIALS


The purpose of this experiment was to test the melting temperature (denature temperature) of the confirmed thermostable griffithsin samples.

Aliquots of each sample were then heated at high temperatures (50,60, 65, 70, 75, 80, 90 degree celcius) for 30 and 60 minutes. The results were analyzed using SDS-PAGE.

Figure 5: Monomer GRFT for 30 minutes

Figure 6: 12% Bis-Tris polyacrylamide gel. Thermostable Dimer Griffithsin for 30 minutes

Figure 7: 12% Bis-Tris polyacrylamide gel. Thermostable Dimer Griffithsin for 60 minutes

Figure 8: 12% Bis-Tris polyacrylamide gel. Thermostable Monomer for 60 minutes

Demonstration of Thermostability


Each thermostable griffithsin sample—monomer and dimer—displayed a band at ~35 kDa, confirming expression of Griffithsin. Furthermore, the band was not present in the empty vector well of any of the gels.

As both dimer and monomeric form were visible on the gel and thus stable until 75 degrees celsius when heated 30 minutes. The more stable dimer form was stable until 90 degrees celsius.

At 60 minutes, both types of Griffithsin were stable until 75 degrees celsius.

Importance


The wildtype melting temperature of Griffithsin is 78 degrees celsius. Our maximum melting temperature of 90 degrees celsius represents a >10 degree change in melting temperature.

Since, the majority of E. coli proteins denature around 60 degrees celsius. Our results indicate that a thermostable version will allow for a heat purification—for up to 30 minutes at 90 degrees celsius—of fermentations to eliminate contaminating E. coli native protein. This allows for a degree of heat purification significantly greater than that using the wild-type Griffithsin.