(48d) Soluble Human Lysozyme Production in Escherichia Coli Via an Engineered Anti-Toxin Switch

The accelerating spread of drug-resistance among bacterial pathogens underlies a critical need for novel antibacterial therapies capable of treating drug-resistant infections while slowing the development of new resistance phenotypes.  Natural bactericidal peptides and proteins represent potential leads in the search for new drugs, and human lysozyme (hLYS) constitutes one particularly promising molecular scaffold. hLYS exerts both catalytic and non-catalytic antibacterial activities, and due in part to this multimodal action hLYS is relatively broad-spectrum, killing both Gram-positive and Gram-negative bacteria.  Moreover, the redesign of hLYS through biomolecular engineering has the potential to yield a diverse panel of antibacterial biocatalysts that are tailored to meet specific clinical needs.  However, efficient development of such therapeutic candidates requires laboratory access to gram-plus scale quantities of material.  While hundreds of kilograms of wild-type hLYS can be produced in recombinant rice, this plant-based system is not readily scaled down to bench top production.  To address this issue, we have recently developed the first expression system capable of producing folded, soluble hLYS in E. coli cells.  Our system harnesses protein folding chaperones and a natural anti-lysozyme protective protein to generate >1000-fold increases in soluble hLYS relative to prior reports in E. coli.  Our original design exploited E. coli’s ease of culture, short doubling time, and facile genetics to yield an expression platform that was moderately scalable and benefited from rapid turnaround.  Critically, the system could be implemented in even the most minimally equipped biotechnology laboratory.  Unfortunately, the overly robust nature of the hLYS-inhibitor interaction reduced the efficiency of purification, which was originally envisioned as a one-step IMAC process.  We have therefore redesigned the natural inhibitor to create a switchable entity that protects the E. coli host during induction but efficiently releases the desired hLYS product upon a mild shift in environmental conditions.  We anticipate that our redesigned expression and purification platform will facilitate further development of engineered lysozymes having utility in disease treatment and other practical applications.