Optimization of expression in Escherichia coli BL21 (DE3), IMAC purification, storage, and preliminary functional characterization of recombinant Chitinase C from Pseudomonas aeruginosa

Chitin is a homopolymer that is found in the abdominal lining and exoskeleton of various insects. Given the increasing environmental pollution due to chemical insecticides used in agriculture, there is great interest in the development of bioinsecticides. Proteins that hydrolyze chitin, like chitinases, have potential in this regard. Pseudomonas aeruginosa PAO1 Chitinase C (ChiC) is a 55 kDa secreted protein, and while the chiC gene has previously been cloned and expressed in Escherichia coli BL21 (DE3), the optimal isopropyl β-D-1- thiogalactopyranoside (IPTG) inducer concentration, incubation time, and temperature for soluble recombinant ChiC (rChiC) expression have yet to be elucidated. We used a factorial experiment to optimize the conditions for soluble rChiC expression, purified soluble rChiC using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin, and conducted a preliminary functional characterization of rChiC using a chitin-binding assay. We found that the soluble protein is optimally expressed under 0.1 mM IPTG induction, 24 hr incubation, and at 20°C. The present study used histidine in place of the more commonly used imidazole for competitive washing and elution in IMAC, illustrating the potential of histidine as a competitive agent for protein purification. Finally, we observed that the purified rChiC has a functional chitin-binding domain, demonstrated by a chitin-resin binding assay. Overall, our study demonstrates the preliminary optimization of expression, purification, and functional characterization of rChiC present in the soluble fraction in E. coli BL21 (DE3). This study reveals potential for future research aimed at further optimization of soluble rChiC and subsequent functional characterization of the chitinolytic domain of rChiC using in vitro and in vivo systems.