Abstract 1748 Purification of Rubredoxin tagged Recombinant Proteins using the Three Phase Partitioning technique

Obstacles persist in producing and purifying recombinant proteins (RCP) and peptides. RCP production of < 50 amino acid peptides is prone to proteolysis, and most recombinant protein ultimately aggregates to inclusion bodies. Purification techniques like conventional chromatography require multiple steps with specialized equipment and demand significant time and labor commitment. Herein, we aim to overcome some of these concerns by utilizing rubredoxin (RD), a 7kDa hyperthermophile protein from Pyrococcus furiosus. RD is stable over various pH, temperature, and denaturants. Fusing it to RCP enhances the recombinant peptide expression making it more soluble thus increasing the protein yield. It has an iron-sulfur cluster embedded in its structure that possesses a red color that aids in the direct detection and quantification of target proteins. Furthermore, we have successfully incorporated three-phase partitioning (TPP) as a purification technique which involves the addition of ammonium sulfate and tert-butanol to the crude extract of RD-peptide forming three layers. We modified our TPP method by adding a high urea concentration to isolate and concentrate Rd-fused peptides in the urea layer while cellular debris salts out in the middle layer. Using this strategy, we purified Rd-fused recombinant peptides from a cell lysate in less than 30 minutes. The combination of the proposed RD tag with the TPP purification technique highlights crucial information to develop an affordable, time-efficient, and simple purification scheme that eliminates the need for specialized instruments and provides higher yields at a relatively low cost for recombinant peptides.