Engineering Stable Protein Nanosheets for In Vivo Applications

Protein nanosheets (pNS) are two-dimensional, self-assembling protein biomaterials with a thickness less than 100 nm. These pNS are made in an aqueous environment under end-over-end rotation using two fusion proteins, a hydrophobic elastin-like protein (ELP) fused between two arginine-rich zippers (Z_R) and a hydrophilic functional globular protein attached to a leucine-rich zipper (Z_E). The mechanism behind the assembly of these pNS is the hydrophobic interaction between the ELPs and the alignment of the hydrophobic and hydrophilic moieties in the assembled structure along the continually expanding and contracting air-water interface due end-to-end rotation. Since the ultimate goal is to use these pNS as scaffolds for surface display of therapeutic proteins for in vivo studies, assessing the stability of the pNS are vital in this respect.

With this in mind, the motivation for this research was engineering the pNS with ELP variants of different lengths and hydrophobicity to determine how they affect the stability of pNS in human pooled serum (HPS). Six different ELP variants were tested: Z_R-ELP, Z_R-ELP₃₅, ZR-ELP₅₀, I₅-Z_R-ELP, Y₅-Z_R-ELP. The more hydrophobic ELPs (I₅ and Y₅) were hypothesized to result in greater hydrophobic interactions between ELPs, the longer ELPs (ELP₃₅ and ELP₅₀) were theorized to maximize inter-ELP interactions, and the removal of one ZR was thought to increase ELP interactions at the air-water interface by reducing steric hinderance.

All pNS were made with a Z_E/Z_R ratio of 1 where the globular protein utilized was green fluorescent protein, sfGFP. Notably, for all hybrid pNS 80% of all ELP population were contributed by Z_R-ELP-Z_R and the remaining 20% by the ELP variants of interest. An initial week-long stability test was conducted on the five hybrid pNS and two pure pNS (Z_R-ELP-Z_R and Z_R-ELP respectively) where they were added to HPS and stored at 37℃. Flow cytometry was run at predetermined time intervals. The forward-scattering was observed to establish the size of pNS after incubation with HPS. The side-scattering did not indicate much variability meaning the pNS were being oriented consistently without altering their configurations or crumpling. The hybrid pNS using Z_R-ELP-Z_R and Z_R-ELP was determined to be the most stable as it was the only sample that maintained a fairly constant size and concentration over the duration.

A subsequent week-long stability test was run to determine if the ratio of Z_R contributors made a difference on the stability of these hybrid pNS. Samples where Z_R-ELP contributed 20%, 50%, and 80% of the Z_R population (with the requisite from Z_R-ELP-Z_R) were incubated with HPS. Irrespective of the ratio, the pNS remained stable for five days after which point they appear to disassociate since the mean forward scattering decreases.

Future scope of this research includes substituting a different fluorescent protein in place of sfGFP and varying the ZR contributors after doing so. Upon determining which configuration of hybrid pNS is most stable, the functional protein can be substituted for a biologically active or specific ligand and ultimately be used as active cell engagers.