Expression of Pseudomonas aeruginosa PAO1 Chitinase C in Escherichia coli BL21 (DE3)

Authors

  • Madalena Cunha Rocha UBC MICB 401
  • Matthew Yap UBC MICB 401
  • Michael Yoon UBC MICB 401

Abstract

Chitin is a rigid natural homopolymer found in the exoskeletons of many insects, including agricultural pests. With the importance of chitin in insect metabolism and protection, there have been attempts to identify and develop chitinolytic insecticidal products. Chitinolytic enzymes are naturally produced by bacterial genera such as Pseudomonas. Here, we examine chitinase C (ChiC), a secreted 55kDa protein encoded by the P. aeruginosa PAO1 gene, chiC. ChiC expression in P. aeruginosa is controlled by quorum sensing, and its mode of secretion remains poorly understood. We hypothesized that transforming an expression vector containing chiC into non-pathogenic E. coli BL21 (DE3) would allow for expression of ChiC, and that purified ChiC would retain its previously characterized chitinolytic and insecticidal activity. We cloned chiC from an established chiC-containing vector (pGKMS21) into pET-28a to create pM3CRYY, a chiC expression plasmid.. Agarose gel electrophoresis confirmed that we amplified chiC from pGKMS21. Next, we ligated chiC between the EcoRI and HindIII restriction sites of pET-28a to create pM3CRYY. Upon confirming the identity of the construct via PCR and Sanger Sequencing, we transformed it into E. coli BL21 for protein expression. SDS-PAGE analysis of IPTG-induced BL21 pellet lysates and supernatant showed that ChiC was expressed after induction, though secretion of the protein did not occur. Ultimately, our work was able to create a ChiC expression system in E. coli, which may be further optimized to study the secretion of ChiC secretion, as well as its intracellular modification and regulation mechanisms.

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Published

2022-08-31