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 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 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 chromatography, 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.

Cloning, Characterization, and Production of a Novel Lysozyme by Different Expression Hosts

“…However, the production of natural lysozymes in bacteria is limited by the inefficient protein expression system. Previous works in E. coli have produced inactive small quantities of enzyme or have been hampered by the isolation of inclusion bodies and refolding, which is inefficient [3,9,16,23]. Several studies have been performed in yeastbased systems and moderate yields have been described in some of these hosts [8,18].…”

“…Hen egg white lysozyme is produced at the metric ton scale by purification from eggs, and recent advances in transgenic plant technologies have led to similarly low production costs for recombinant human lysozyme [31]. Human lysozyme has also been expressed in Escherichia coli [16], Saccharomyces cerevisiae [12], and Pichia pastoris [28].…”

Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection

“…Fueled in part by advances in recombinant protein production technologies (33,34), endogenous antimicrobial proteins, such as lysozyme, have emerged as prospective therapeutic candidates. These agents are naturally occurring within humans, play important roles in innate immunity, exert broad-spectrum antimicrobial activity, and function via mechanisms distinct from those of traditional antibiotics.…”

Genetically Enhanced Lysozyme Evades a Pathogen Derived Inhibitory Protein

“…Genes coding for Ivyc and E. coli MliC were amplified from E. coli JM105, and that for Ivyp from P. aeruginosa strain PA01. Expression vectors pET26b-Ivyc-his, pET26b-Ivyp-his, and pET26b-MliC-his were constructed and subsequently transformed into Shuffle® T7 Express cells (New England Biolabs, USA) and produced as described previously (42). …”