Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system

Abstract

Antibacterial factor 2 (ABF-2) is a 67-residue antimicrobial peptide derived from the nematode Caenorhabditis elegans. Although it has been reported that ABF-2 exerts in vitro microbicidal activity against a range of bacteria and fungi, the structure of ABF-2 has not yet been solved. To enable structural studies of ABF-2 by NMR spectroscopy, a large amount of isotopically labeled ABF-2 is essential. However, the direct expression of ABF-2 in Escherichia coli is difficult to achieve due to its instability. Therefore, we applied a coexpression method to the production of ABF-2 in order to enhance the inclusion body formation of ABF-2. The inclusion body formation of ABF-2 was vastly enhanced by coexpression of aggregation-prone proteins (partner proteins). By using this method, we succeeded in obtaining milligram quantities of active, correctly folded ABF-2. In addition, 15 N-labeled ABF-2 and a well-dispersed heteronuclear single quantum coherence (HSQC) spectrum were also obtained successfully. Moreover, the effect of the charge of the partner protein on the inclusion body formation of ABF-2 in this method was investigated by using four structurally homologous proteins. We concluded that a partner protein of opposite charge enhanced the formation of an inclusion body of the target peptide efficiently.

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

“…These genes have been named antibacterial factor (abf) genes in the nematode. ABF-2 has been characterized at the peptide-level in two studies [74,75]. The heterologously expressed peptide showed high in vitro activity against a diversity of microbes, ranging from Gram-negative to Gram-positive bacteria and yeasts.…”

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 .…”

“…Tomisawa et al succeeded in expressing a large amount of cysteine-rich AMPs such as antibacterial factor-2 and the mouse a-defensin, cryptidin-4 in E. coli cells as inclusion body by coexpression of aggregation-prone partner protein. 30,31 Saito et al reported the enhanced expression of somatomedin C by coexpression of insulin-like growth factor I. 37 Similarly a potent AMP, buforin IIb, was successfully produced by coexpression of human gamma interferon.…”

“…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 . Thus, negatively charged aggregation‐prone partner protein can effectively enhance the inclusion body formation of positively charged AMPs.…”

“…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. 30 Thus, negatively charged aggregation-prone partner protein can effectively enhance the inclusion body formation of positively charged AMPs. To construct a coexpression system, we utilized a commercially available pCOLADuet1 vector (Novagen) as coexpression vector.…”