Pseudomonas aeruginosa is a bacterium that produces elastase, a proteolytic enzyme. Elastase disrupts the tight junctions of epithelial cells in the lungs, so in Cystic Fibrosis patients it can cause acute and further chronic infections that potentially spread, leading to increased medical complications. This study represents a valid protocol and experimental design to express elastase using the plasmid-cloning vector PET28b and the purification of elastase in E. coli. Genomic DNA was isolated from P. aeruginosa (PAO1, ATCC) and selected regions were amplified using PCR with lasB gene specification. Amplified DNA was ligated into the plasmid PET28b and E. coli were transformed to support the recombinant vector and produce a viable elastase product. The recombinant DNA was sequenced and accurately matched the pro-elastase sequence (1497 bp in length). Purification of enzyme produced via heterologous expression in Clear Coli LPS free E. coli was aided via NTA-Ni Liquid Chromatography system by the incorporation of a 6xHistidine affinity tag engineered on the C-terminus of elastase. Analysis by SDS-PAGE revealed a product of ~34 kDa, consistent with the molecular weight of the naturally produced mature elastase. Proteolytic activity was determined via Skim Milk Agar diffusion displaying an active recombinant product and activity determined using Elastin-Congo Red substrate.

Cloning and expression of Pseudomonas aeruginosa elastase in E. coli for examining inflammatory response of lung tissue

253rd National Meeting (2017)

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