[Purification of recombinant proteins with silkworm chitin-binding protein CPAP3 as a protein tag]

There are various chitin-binding proteins in insects. Among them, the CPAP3 protein family containing the chitin-binding domain ChtBD2 is one of the most abundant types in the epidermis. This study aims to use insect chitin-binding proteins as tag proteins to explore their application potential in the field of protein purification, and simultaneously investigate the relationship between the number of domains of CPAP3 protein and its binding capacity. CPAP3-A (abbreviated as CPAP3) from silkworm was used as the tag protein, and enhanced green fluorescent protein (EGFP) as the target protein, to achieve the fusion expression of the insect epidermal protein and EGFP. With chitin/chitosan materials as the stationary phase, the application potential of CPAP3 in protein purification was explored through binding experiments. The results showed that the fusion protein CPAP3-EGFP could bind to four different types of chitin/chitosan. Orthogonal experiment analysis optimized the reaction conditions as follows: β-chitin as the matrix and binding buffer at pH 6.5, under which the adsorption rate reached 70.44%. To further investigate the relationship between the number of domains and binding capacity, we conducted truncation treatment on CPAP3. The results indicated that the protein containing three domains had significantly higher binding capacity than the other two truncated groups, suggesting a positive correlation between the binding capacity of the protein tag and the number of domains. To effectively release the target EGFP bound to chitin, we introduced a TEV protease cleavage site between the target protein and the tag protein. Experimental results showed that after digestion with TEV protease, the protein sample exhibited a new band at 28 kDa, confirming that the fusion protein could be cleaved by TEV protease and the target protein was released into the supernatant. This study provides references for the application of insect chitin-binding proteins as affinity tags.