Refolding of Recombinant Chitinase C of Pseudomonas aeruginosa purified from insoluble fractions using IMAC purification

Abstract

Chitinase C (ChiC) from Pseudomonas aeruginosa can potentially be developed into a bioinsecticide agent, as it can break down chitin, a crucial component of most insect exoskeletons. However, due to the pathogenic potential of P. aeruginosa, developing ChiC for use in agricultural industries is not desirable. To express recombinant ChiC (rChiC), previous studies transformed Escherichia coli BL21 with pM3CRYY, a pET-28ɑ plasmid containing the chiC gene. Although they observed the expression of rChiC in E. coli, they did not go further to purify rChiC. The aim of this study was to obtain purified and functional rChiC. We investigated the yield of rChiC expression under different sets of conditions in E. coli BL21 transformed with pM3CRYY, followed by purification and refolding of rChiC. The conditions that led to the highest yield of expressed rChiC were found to be 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) concentration with a 2 hrs induction period at 37 °C. At these conditions, the majority of the expressed rChiC was insoluble. The rChiC was purified from the insoluble fraction of the cell lysates using immobilized metal (Ni²⁺) affinity chromatography under denaturing conditions. Subsequently, we refolded the enzyme through dialysis, which yielded 11.7 g of rChiC per 1 L of E. coli culture. The unique pattern of cleavage in limited proteolysis demonstrated that the purified rChiC was refolded into a uniform tertiary structure in dialysis. The successful purification of the refolded rChiC using a chitin-binding column suggested that the enzyme binds to the chitin beads. This suggests that the binding domain of the refolded rChiC is functional. The overall results indicate functional rChiC can be extracted from insoluble fraction of cell lysates with a high yield in a time and cost-efficient manner, which paves the way for future biotechnological applications of ChiC in agricultural industries.