Development of a Chitin-Based Purification System Utilizing Chitin-Binding Domain and Tobacco Etch Virus Protease Cleavage for Efficient Recombinant Protein Recovery

This study aims to develop an efficient chitin-based purification system, leveraging a novel design where the target proteins, superfolding green fluorescent protein (sfGFP) and <i>Thermus antranikianii</i> trehalose synthase (TaTS), fused with a chitin-binding domain (ChBD) from <i>Bacillus circulans</i> WL-12 chitinase A1 and a tobacco etch virus protease (TEVp) cleavage site. This configuration allows for the effective immobilization of the target proteins on chitin beads, facilitating the removal of endogenous proteins. A mutant TEVp, H-TEVS219V-ChBD, fused with the His-tag and ChBD, is employed to cleave the target proteins from the chitin beads specifically. Subsequently, fresh chitin beads are added for adsorption to remove H-TEVS219V-ChBD in the solution, thereby significantly improving the purity of the target protein. Our results confirm that this system can efficiently and specifically purify and recover sfGFP and TaTS, achieving electrophoretic-grade purity exceeding 90%. This system holds significant potential for industrial production and other applications.