A novel bi-domain plant defensin MtDef5 with potent broad-spectrum antifungal activity binds to multiple phospholipids and forms oligomers
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| Peptide Synthesis |
Te codon-optimized synthetic genes encoding
MtDef5 and MtDef5B were obtained from GenScript Corporation (Piscataway, NJ) and subsequently cloned
into pPICZαA, as described below. MtDef5 and MtDef5B genes were cloned into the EcoRV site of pUC57. A
sequence encoding the XhoI restriction endonuclease and the KEX2 protease site (CTCGAGAAAAGA) was
added upstream of the mature coding sequence of each defensin. Two stop codons, along with an XbaI restriction enzyme site (TAGTAATCTAGA) were added downstream of the mature coding sequence. Synthetic genes encoding
the MtDef5 γ-core motif variants were custom synthesized by GenScript (Piscataway, NJ). Te synthetic
genes encoding MtDef5H36A,R37A/H93A,R94A (MtDef5_V1), MtDef5Q38A, G39A/Q95A, G96A (MtDef5_V2), MtDef5F40A,G41A/
I97A,G98A (MtDef5_V3) and MtDef5F42A/F99A (MtDef5_V4) were thus generated.Te MtDef5-phospholipid interactions
were detected using the affinity-purified MtDef5-derived peptide (CQKRSTTWSGP) polyclonal antibody
(GenScript, Piscataway, NJ) and HRP-conjugated Goat Anti-Rabbit IgG secondary antibody following the manufacturer’s
protocol with minor modifcations as described previously14. |
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