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

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.