Construction of plasmid pRMGS22 for the expression and His-tag purification of recombinant chitinase encoded by the chiC gene of Pseudomonas aeruginosa PAO1

Abstract

The use of chemical pesticides has been the leading approach in agriculture to protect crops from pest and insect-induced damage. However, environmental pollution and human health problems have resulted from excessive chemical pesticide use. As such, there is an urgent need to explore other pesticides that are more ecologically friendly and sustainable. Chitinase, or ChiC, is a naturally occurring enzyme found in Pseudomonas aeruginosa capable of degrading chitin, a structural component of the insect exoskeleton. Previous studies have investigated the expression of the chiC gene for its potential use as an alternative insecticide. A study by Bodykevich et al. was able to amplify the chiC gene from P. aeruginosa PAO1 and ligate it into the pCR 2.1 vector, forming the pGKMS21 plasmid. In this study, we sought to subclone the chiC gene from the pGKMS21 storage vector into the pET-28a expression vector and transform it into Escherichia coli strain BL21-(DE3). The chiC gene was amplified from the pGKMS21 vector using gradient PCR and was subsequently ligated into pET-28a, forming the pRMGS22 plasmid. The pRMGS22 plasmid was further propagated in E. coli strain DH5⍺, isolated, and transformed into E. coli strain BL21-(DE3). The resulting clones of the pRMGS22 plasmid were analyzed through Sanger Sequencing. The nucleotide sequence and the transcribed protein sequence confirmed the presence of chiC from P. aeruginosa PAO1 with 100% identity observed using the NCBI BLASTn and BLASTp tools. Future studies will be able to use the pRMGS22 construct to test protein expression and potentially purify the ChiC protein for more in-depth characterization and testing for enzymatic activity. This will allow for further study of the ChiC protein as a potential alternative to chemical pesticides.