Antifungal activity of a recombinant defensin CADEF1 produced by Escherichia coli

Abstract

The defensin CADEF1 has been known to be uptranscripted in the pepper Capsicum annuum L., upon bacterial attack, abiotic elicitors and environmental stresses. However, the native form of CADEF1 has not been purified from the pepper, and its activity against fungi has been uncertain. In this study, the full-length cDNA of CADEF1 was obtained by fragment piecing-together method. Thereafter, its mature peptide coding sequence was inserted into bacterial expression vector pET28a(?), and the recombinant vector was transformed into Escherichia coli BL21 (DE3). Based on SDS-PAGE and Western blotting analysis, the recombinant CADEF1 was produced as a 5.6 kDa protein in the form of inclusion body in E. coli BL21 (DE3) and the expression level accounted for 15% of the bacterial total protein. Further processing through protein refolding and purification, converted the inclusion body type of CADEF1 into active form with the yield about 13 lg/ml of culture. The refolded CADEF1 significantly showed the activity against growth of the fungal pathogen Verticillium dahliae on average by 67.9% in vitro assay. These results verified the novel activity of CADEF1, which might benefit in prevention of the fungus-related disease in agricultural production.

Cysteine-stabilized αβ defensins: From a common fold to antibacterial activity

“…The heterologous expression of AMPs also faces intracellular degradation by proteases and their negative activities against host cells [93]. Several antifungal defensins were expressed in Escherichia coli and recovered to their native form through diverse purification methods and a renaturation process [17,58,61]. In contrast, antibacterial defensins present a clear limitation in their expression in E. coli, due to the deleterious effect that they could produce on this host organism.…”

Molecular cloning and characterization of a subtilisin-like protease from Arabidopsis thaliana

“…The AtSBT1.9 protein was expressed using a prokaryotic system for a number of reasons. Compared with other expression systems, E. coli serves as an excellent host for recombinant protein production because it provides an economical and fast way to produce the molecules in relatively large amounts, although yields of correctly folded and functional protein can be low because of protein aggregation (Li and Li, 2009). However, the pMAL protein fusion system provides a convenient method for directing fusion proteins to the periplasm where they are allowed to fold.…”

A review of a promising therapeutic and agronomical alternative: Antimicrobial peptides from Capsicum sp.

“…In the case of animals, strong evidence supporting this hypothesis comes from the expression of C. chinense defensin in the epithelial tissues of bovine and whose expression protected the tissues against C. albicans (Anaya-López et al, 2006). Li and Li (2009) also obtained additional evidence with transcript of defensine CDef1 from C. annuum. The full length cDNA of CDef1 was expressed in Escherichia coli by using the expression vector pET28a.…”

“…Heterologous expression of plant defensin could be used as a strategy to confer protection to the receptor orga-nisms against some common infectious agents. In the case of C. annuum defensin CADEF1, it is well established that defensin transcript is up- regulated upon bacterial attack, abiotic elicitors and environmental stresses; unfortunately, the complete transcript of the native protein have been elusive to be cloned or purified from chili pepper (Li and Li, 2009). In a recent study using a cDNA library constructed from ripening C. annuum fruits, it was described that the isolation of full length cDNA of 534 bp that encodes a defensin denominated CDef1.…”

“…The full length cDNA of CDef1 was expressed in Escherichia coli by using the expression vector pET28a. Western blot of the recombinant suggests that activity of CDef1 might benefit by preventing fungus-related diseases in agricultural production (Li and Li, 2009). In an additional study, Zainal et al (2009) generated transgenic tomatoes carrying C. annuum defensin gene Cdef1 which encodes a 5 kDa peptide.…”