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

Date Published: August 15, 2013

Publisher: Springer

Author(s): Satoshi Tomisawa, Eri Hojo, Yoshitaka Umetsu, Shinya Ohki, Yusuke Kato, Mitsuhiro Miyazawa, Mineyuki Mizuguchi, Masakatsu Kamiya, Yasuhiro Kumaki, Takashi Kikukawa, Keiichi Kawano, Makoto Demura, Tomoyasu Aizawa.


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.

Partial Text

Antimicrobial peptides play an important role in innate immunity as a part of the host defense response (Ganz 2003; Radek and Gallo 2007). Antimicrobial peptides are thought to kill bacteria by breaking their cell membranes, although the exact mechanisms are still unclear (Sato and Feix 2006; Mani et al. 2005). To date, numerous antimicrobial peptides have been identified in a wide range of organisms, such as mammals, insects, and plants (Zasloff 2002).

Because the correct disulfide arrangements of disulfide-containing peptides are difficult to obtain, the yeast P. pastris is widely used to express peptides containing intramolecular disulfide bridges. The main advantage of P. pastris as a host is that it is expected to secrete peptides with correct disulfide bridges directly into culture medium (Daly and Hearn 2005). Several CSαβ-type antimicrobial peptides have been produced by using P. pastris, and these were well characterized (Wang et al. 2008; Zhang et al. 2011; Wiens et al. 2011). However, a previous study showed that P. pastris is not a suitable host for large-scale production of ABF-2. Although the antimicrobial spectrum of ABF-2 was investigated, structural and mutational studies of ABF-2 have not yet been conducted because of the low yield of ABF-2 in P. pastris (Kato et al. 2002). Therefore, it is very important to develop an alternative expression method that enhances the expression level of ABF-2.

The authors declare that they have no competing interests.




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