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.
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
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. 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. Previous studies in our lab produced the following SDS-PAGE gel confirming that Wildtype-Griffithsin runs at 12 kDa. 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. IPTG INDUCED EXPRESSION
Figure: Homogenization
CONFIRMATION OF GRIFFITHSIN EXPRESSION
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
Figure 2: From a previous experiment.
Figure 3: Thermostability