Extraction of Zera® fusion proteins in aqueous two-phase systems

The N-terminal proline-rich domain (Zera^®) of the maize storage protein γ-zein is a self-assembling domain produced and patented by EraBiotech, which can be fused with proteins of interest. Aqueous two-phase system (ATPS) is a biocompatible method for protein purification, without denaturation or loss of biological activity due to the high water content, low interfacial tension and stabilizing effect of the polymers. This work aims at developing and optimizing a scalable process for the extraction of Zera^® fusion model proteins, Zera^®, Zera^®-alpha amylase and Zera^®-lipase produced in Bacillus brevis to predict the partition of high-value recombinant proteins, as Zera^®-Prostatic Acid Phosphatase (PAP), in PEG-phosphate ATPS. The influence of Polyethylene Glycol (PEG) molecular weight (MW), tie line Length (TLL), pH, sample load and neutral salt addition were evaluated and optimized for each recombinant proteins. Based on the partition results and the specific proteins characteristics (hydrophobicity and MW), a system with low TLL, PEG MW between 6 000 and 8 000, pH between 8-9 and loading percentage between 20 and 30% should probably be a good system for Zera^®-PAP selective extraction.