Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T,). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T-t such that purification conditions (T-t) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T-t of an ELP. A negative correlation between T-t and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendainistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T-t by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.