iFLOAT: Isolation of Floater-Linked Overexpressed-protein Ascender Technology

iFLOAT is our attempt to develop a novel protein purification strategy using cyanobacterial gas vesicle tagged proteins. Our project strives to reduce downstream recombinant protein purification costs and will vastly improve cost-effectiveness and productivity of industries reliant on recombinant protein production.

To facilitate this, we are engineering E. coli to express gas vesicle proteins (gvpA and gvpC) from cyanobacteria (Planktothrix rubescens). The recombinant protein of interest will be fused to gvpC using an amino acid linker comprising the sequence specific to the enzyme TEV protease (EDLYFQ|S).

When the cells have overexpressed enough of the recombinant protein, we will lyse the cells and obtain a protein extract. Since the recombinant protein of interest is fused to a gas vesicle, it will float to the surface of the protein extract and can be skimmed off. After the desired level of purity is obtained (by repeated resuspension and skimming), TEV protease can be added to cleave the recombinant protein of interest from the gas vesicle, dispersing it into the solution while the pure gas vesicles remain at the surface.

For our project, we will demonstrate a proof of concept using the recombinant protein sfGFP, which we will tag to gvpC. Following this, we will demonstrate the same system in Pichia pastoris to show that eukaryotic protein purification can also be simplified by our method.