Difference between revisions of "Part:BBa K2959008"

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Expressible Wheat antimicrobial peptide 1b with Erv1p

Since WAMP1b is a peptide with five disulfide bonds, a post-traductional modification hard to replicate on prokaryotic expression systems, the construct allows for the co-expression of the peptide with a truncated version of Erv1p. This is a protein from Saccharomyces cerevisiae capable of catalyzing the formation of disulfide bonds.
This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for WAMP1b as a fusion protein with a 6x His-Tag, another ribosome binding site, the coding sequence for Erv1p, and a double terminator. This construct allows the expression of WAMP1b, an antifungal peptide from Triticum kiharae seeds, in E. coli, as well as the protein Erv1p. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The part is designed to code for a fusion protein of WAMP1b with a polyhistidine tag (6x His-Tag) at its N-terminus for purification by immobilized metal affinity chromatography.

Usage and Biology

WAMP1b is an antimicrobial peptide from Triticum kiharae seeds. This peptide consists of 116 amino acids with a molecular weight of 11.5 kDa. Among these aminoacids, 10 cysteines are included, which form 5 nonconsecutive disulfide bonds C38-C53, C47-C59, C50-C78, C52-C66, and C71-C75. This composition makes it a highly stable molecule.^{1, 2}

Hevein-like peptides, such as WAMP1b, contain a chitin binding domain as a structural motif of 35 amino acids with specific cysteine and glycine residues. Given this, the peptide is able to successfully bind to chitin, acting as a plant defense mechanism against fungi and certain insects.²

WAMP1b also inhibits fungalysin Fv-cmp, a protease produced by Fusarium fungi as a counterattack mechanism to plant’s defenses, that cleaves chitinases. By inhibiting this protease, WAMP1b acts as an additional defense mechanisms against fungal pathogens. It has also been observed that this peptide directly inhibits hyphal elongation.³

In an investigation done by Slavokhotova et al., (2014), WAMP1b was tested against various fungi, and it was proved effective against against F. verticillioides with an IC50 of 2.7 µg/ml.

For a successful expression in E.coli, it is indispensable to avoid interactions that result in an aggregation of folding intermediates. Disulfide bonds can be involved in the structural, catalytic and signaling roles of the protein, but the formation of disulfide bonds can have certain problems that can cause misfolding, aggregation and low yields during the production of the protein⁴. The usage of Erv1p has been proposed as a mechanism for the formation of disulfide bonds in recombinant proteins in prokaryotic expression systems. It has shown that, thanks to its ability to form disulfide bonds de novo, co-expression of Erv1p allows the proper formation of disulfide bonded proteins in the cytoplasm of E. coli.⁵

The ERV1 gene from Saccharomyces cerevisiae encodes for a 189 amino acids protein, Erv1p, that is involved in different processes of mitochondrial biogenesis and maintenance^{5, 6}. The protein shows a flavin-linked sulfhydryl oxidase enzymatic activity linked to the carboxy-terminal domain. Thus, Erv1p is capable of oxidizing thiol groups in proteins and catalyzing disulfide bond formation. A 15 kDa truncated version of Erv1p consisting of the 117 amino acid residues carboxy-terminal domain shows a similar or improved sulfhydryl oxidase activity compared to the full length protein.⁶

Sequence and Features

References

1.Dubovskii, P. V., Vassilevski, A. A., Slavokhotova, A. A., Odintsova, T. I., Grishin, E. V., Egorov, T. A., & Arseniev, A. S. (2011). Solution structure of a defense peptide from wheat with a 10-cysteine motif. Biochemical and Biophysical Research Communications, 411(1), 14–18. doi: 10.1016/j.bbrc.2011.06.058
2. Odintsova, T. I., Vassilevski, A. A., Slavokhotova, A. A., Musolyamov, A. K., Finkina, E. I., Khadeeva, N. V., … Egorov, T. A. (2009). A novel antifungal hevein-type peptide from Triticum kiharaeseeds with a unique 10-cysteine motif. FEBS Journal, 276(15), 4266–4275. doi: 10.1111/j.1742-4658.2009.07135.x
3. Slavokhotova, A. A., Naumann, T. A., Price, N. P. J., Rogozhin, E. A., Andreev, Y. A., Vassilevski, A. A., & Odintsova, T. I. (2014). Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases. FEBS Journal, 281(20), 4754–4764. doi: 10.1111/febs.13015
4. Veggiani, G., & de Marco, A. (2011). Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase. Protein expression and purification, 79(1), 111-114. doi: 10.1016/j.pep.2011.03.005
5. Hatahet, F., Nguyen, V. D., Salo, K. E., & Ruddock, L. W. (2010). Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microbial cell factories, 9(1), 67. doi: 10.1186/1475-2859-9-67
6. Lee, J. E., Hofhaus, G., & Lisowsky, T. (2000). Erv1p from Saccharomyces cerevisiae is a FAD‐linked sulfhydryl oxidase. FEBS letters, 477(1-2), 62-66. doi: 10.1016/s0014-5793(00)01767-1