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

Abstract

Snakin-1 (SN-1) is a cysteine-rich plant antimicrobial peptide and the first purified member of the snakin family. SN-1 shows potent activity against a wide range of microorganisms, and thus has great biotechnological potential as an antimicrobial agent. Here, we produced recombinant SN-1 in Escherichia coli by a previously developed coexpression method using an aggregation-prone partner protein. Our goal was to increase the productivity of SN-1 via the enhanced formation of insoluble inclusion bodies in E. coli cells. The yield of SN-1 by the coexpression method was better than that by direct expression in E. coli cells. After refolding and purification, we obtained several milligrams of functionally active SN-1, the identity of which was verified by MALDI-TOF MS and NMR studies. The purified recombinant SN-1 showed effective antimicrobial activity against test organisms. Our studies indicate that the coexpression method using an aggregation-prone partner protein can serve as a suitable expression system for the efficient production of functionally active SN-1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1520-1528, 2017.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation

“…It is thought that AMPs which require disulfide cross-linking to form their stable conformation are often degraded because of their instability when expressed in a reducing intracellular environment, thus resulting in a low level of production. Therefore, we tested our previously developed expression system [32][33][34] that promotes inclusion body formation by co-expression of AMPs with HLA, which has a high propensity for aggregation (Fig. 3a).…”

“…However, because of the high solubility of AMPs as a result of their high positive charge, simple expression often makes it difficult to form inclusion bodies of them. To solve this limitation, we developed a method in which AMPs are successfully co-expressed as inclusion bodies with aggregation-prone and negatively charged human α-lactalbumin (HLA) [32][33][34]. The electrostatic and hydrophobic interactions between AMPs and HLA are presumably responsible for the enhanced inclusion body formation.…”

A Redox-Based Autoinduction Strategy to Facilitate Expression of 5xCys-Tagged Proteins for Electrobiofabrication

“…Although it is unknown how the 5xCys tag would affect the folding of the fused protein, our results suggest that a more oxidized environment is preferred for the expression of 5xCys-tagged proteins, which helps to increase soluble target protein content and reduce inclusion body formation. By simply shutting off the oxidative stress response, perhaps this method could benefit the production of not only other Cys-tagged proteins, but other redox-sensitive proteins that require correct disulfide bond formation, such as single chain Fv antibodies (Zhang et al, 2002), antimicrobial peptide snakin-1 (Kuddus et al, 2017), and murine Wnt-1 (Mursula et al, 2006). Again, additional investigations would help us to understand the favored tertiary and quaternary structures of the 5xCys-tagged proteins, for example, how easily do they aggregate and form oligomers?…”