Optimization of Chitinase C expression from the pM3CRYY plasmid in Escherichia coli BL21(DE3) and determination of secretion status

Abstract

Chitin is an essential polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons that can be broken down by chitin-degrading enzymes called chitinases. As conventional pesticides contain chemicals associated with a number of severe health risks, chitinase C (ChiC) expressed from the soil microbe Pseudomonas aeruginosa has been proposed as a safer and more sustainable alternative to target chitin-containing pests. A previous study generated a ChiC expression plasmid, pM3CRYY, by cloning chiC from P. aeruginosa PAO1 into a pET28a backbone. Through induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), they demonstrated expression of a 55 kDa protein from pM3CRYY, though the identity of this protein was not confirmed. It is also unknown if the expressed protein is secreted when expressed in E. coli BL21. Thus, we aimed to confirm the identity of 6xHis-tag conjugated ChiC off pM3CRYY, and to determine the optimal IPTG induction conditions for ChiC expression. We found that chiC in pM3CRYY is conjugated to a 6xHis-tag at the 5’ region, and confirmed the presence of His-tagged ChiC in IPTG-induced pM3CRYY Escherichia coli BL21(DE3) cultures. Optimal conditions for protein expression were 0.1 mM IPTG for >6 hours at 37ºC. Additionally, lysing cells by bead beating resulted in higher amounts of protein extracted compared to a boiling lysis method. Interestingly, we also detected 6xHis-tagged ChiC in the soluble fractions of induced cultures. However, it was unclear if the protein was being released into the extracellular space because of active secretion or through the action of cells lysing and releasing their intracellular contents. Therefore, we attempted to determine if ChiC was being secreted under induction conditions by probing for the presence of the putative cytosolic chaperone protein, DnaK. Although our results suggest cytosolic proteins and ChiC are released into culture supernatants as a result of cell lysis, this experiment should be repeated before concluding the secretion status of ChiC in this expression system.