Abstract

Pseudomonas aeruginosa causes acute nosocomial as well as community acquired pneumonia. In cystic fibrosis patients, this organism causes recurrent infections and inflammation with a high rate of morbidity and mortality. To support research in elucidating the molecular mechanisms of Pseudomonas elastase (PE) on disease progression, PE was expressed in E. coli and the protein purified for further application. This study represents a valid experimental protocol to produce. Genomic DNA was isolated from P. aeruginosa and selected regions were amplified using PCR with lasB gene specification. Amplified DNA was ligated into plasmids 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 ClearColi LPS free E. coli was aided via Ni-NTA Liquid Chromatography system by the incorporation of a 6x Histidine affinity tag engineered on the C-terminus of elastase. Analysis by SDS-PAGE revealed a product of ~34 kDa in molecular weight consistent with the molecular weight of the naturally produced mature Elastase. Proteolytic activity was determined via Skim Milk Agar diffusion. Elastolytic activity was determined by Elastin-Congo Red substrate to be two times the activity of the commercial enzyme. Cellular anatomy and physiology experiments confirmed the conserved pathologic effects of the Recombinant Elastase. Immunofluorescent study demonstrated preliminary data that Recombinant Elastase had the ability to disrupts tight junction structure as evidenced by fragmentation of ZO-1 proteins in Calu-3 cell monolayers.

Date of publication

Spring 4-27-2017

Document Type

Thesis

Language

english

Persistent identifier

http://hdl.handle.net/10950/561

Committee members

Ali Azghani, Dustin Patterson, Brent Bill

Degree

Master of Science in Biology

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