Efficient production of a correctly folded mouse α-defensin, cryptdin-4, by refolding during inclusion body solubilization

Abstract

Mammalian α-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of α-defensins, large amounts of α-defensins are essential. Although many expression systems for the production of recombinant α-defensins have been developed, attempts to obtain large amounts of α-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse α-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in E. coli expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies.Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of α-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step.3

Enhanced expression of cysteine‐rich antimicrobial peptide snakin‐1 in Escherichia coli using an aggregation‐prone protein coexpression system

“…In some studies, the coexpression of an insoluble partner protein has been reported to enhance the inclusion body formation of the target peptide. Tomisawa et al succeeded in expressing a large amount of cysteine‐rich AMPs such as antibacterial factor‐2 and the mouse α‐defensin, cryptidin‐4 in E. coli cells as inclusion body by coexpression of aggregation‐prone partner protein . Saito et al reported the enhanced expression of somatomedin C by coexpression of insulin‐like growth factor I .…”

“…Recently we developed a coexpression method that enhanced the inclusion body formation of the target peptide by coexpression of an aggregation‐prone protein as a partner protein. The charge of the partner protein was previously shown to affect the inclusion body formation by the target peptide, with an oppositely charged partner protein being considered most suitable for efficient formation of inclusion bodies of the target peptide .…”

Perspectives for clinical use of engineered human host defense antimicrobial peptides

“…The most commonly used host cell has been Escherichia coli mainly because it grows easily, fast and is inexpensive, and also because it is a well-established expression system (Pazgier and Lubkowski 2006; Tomisawa et al. 2015). Disadvantages of the use of these recombinant strategies to produce HDPs are their potential toxicity to the host and their susceptibility to proteolytic degradation (Li 2009).…”

“…The formation of inclusion bodies as a novel way to prevent degradation and improve productivity has also been assayed with promising results compared to the conventional method (Tomisawa et al. 2015). …”

“…2014; Tomisawa et al. 2015). Consequently, the synthesis and evaluation of HDPs-derivatives, -analogs or -mimetics have already been reported with encouraging results, regarding their antimicrobial activity and feasible synthesis (Sigurdardottir et al.…”