Purification of Chitinase C in transformed E. coli BL21 (DE3) preserves a functional Chitin-binding Domain

Abstract

Conventional pesticides play an integral role in commercial agriculture, however, many of these pesticides also have a negative impact on human health, leading to increased interest in finding safe alternatives. Chitin, a biopolymer commonly found in the exoskeleton of insects, has received significant attention as a potential target for the development of biopesticides. Chitinolytic enzymes are naturally produced by many bacterial species such as Pseudomonas aeruginosa. In particular, the 55kDa chitinolytic enzyme chitinase C (ChiC), secreted by Pseudomonas aeruginosa PAO1, has demonstrated insecticidal activities in previous studies. ChiC has been extensively characterized in its native host and previously transformed into other bacteria such as Escherichia coli using the pM3CRYY chiC expression plasmid to isolate ChiC for purification and mechanistic studies. We hypothesized that soluble protein purification of ChiC would allow for the preservation of a functional chitin-binding domain. Purification was performed using immobilized metal affinity chromatography and dialysis, and the activity of the chitin-binding domain in purified ChiC was tested using chitin resin affinity purification. The successful purification of ChiC with a functional chitin-binding domain highlights the potential of the ChiC expression system in E. coli BL21(DE3), which may be further investigated to elucidate the possible functionality of the whole protein.