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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23553 43452 2013 8 PDF Available
Title
Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme
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.

► First report of Ivy and lysozyme co-expression in Escherichia coli. ► Ivy sequesters lysozyme activity and protects host cells from lysis. ► His-tagged Ivy can be leveraged for downstream 1-step purification of lysozyme. ► Use of molecular chaperones increased soluble lysozyme expression. ► Soluble lysozyme expression was improved by 3000-fold over previous reports.

Keywords
Lysozyme; Ivy; Inhibitor of vertebrate lysozyme; Molecular chaperones; Escherichia coli; Bioreactor; Soluble expression
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Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 164, Issue 1, 10 March 2013, Pages 1–8
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us