Engineering Escherichia coli for Soluble Expression and Single Step Purification of Active Human Lysozyme.

Thayer School of Engineering.
Journal of Biotechnology (Impact Factor: 2.87). 12/2012; 164(1). DOI: 10.1016/j.jbiotec.2012.11.007
Source: PubMed


Genetically engineered variants of human lysozyme represent promising leads in the battle against drug-resistant bacterial pathogens, but early stage development and testing of novel lysozyme variants is constrained by the lack of a robust, scalable and facile expression system. While wild type human lysozyme is reportedly produced at 50-80kg per hectare of land in recombinant rice, this plant-based system is not readily scaled down to bench top production, and it is therefore not suitable for development and characterization of novel lysozyme variants. Here, we describe a novel and efficient expression system capable of producing folded, soluble and functional human lysozyme in E. coli cells. To achieve this goal, we simultaneously co-express multiple protein folding chaperones as well as harness the lysozyme inhibitory protein, Ivy. Our strategy exploits E. coli's ease of culture, short doubling time, and facile genetics to yield upwards of 30mg/L of soluble lysozyme in a bioreactor system, a 3000-fold improvement over prior efforts in E. coli. Additionally, molecular interactions between lysozyme and a his-tagged Ivy allows for one-step purification by IMAC chromatography, yielding as much as 21mg/L of purified enzyme. We anticipate that our expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.

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    ABSTRACT: The spread of drug-resistant bacterial pathogens a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature's repertoire of bactericidal proteins and peptides. In humans, the airway-surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme's dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.
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