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J Biotechnol. 2013 Mar 10;164(1):1-8. doi: 10.1016/j.jbiotec.2012.11.007. Epub 2012 Dec 7.

Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme.

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  • 1Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA.

Abstract

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 50–80 kg 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 Escherichia 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 30 mg/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, yielding as much as 21 mg/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.

Copyright © 2012 Elsevier B.V. All rights reserved.

PMID:
23220215
[PubMed - indexed for MEDLINE]
PMCID:
PMC3594478
Free PMC Article
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