Expression of plectasin in Bacillus subtilis using SUMO technology by a maltose-inducible vector

Abstract

Plectasin, the first fungus defensin, is especially efficient against Gram-positive bacteria. To explore an effective approach for expressing plectasin in Bacillus subtilis, the sequence encoding plectasin fused with the small ubiquitin-like modifier (SUMO) gene, the 6 × His gene and the signal peptide of SacB were cloned into an E. coli-B. subtilis shuttle vector pGJ148 in which the maltose utilization operon promoter Pglv directed the expression. The fusion protein successfully secreted in culture and approximately, 41 mg of the recombinant fusion protein SUMO-plectasin was purified per liter of culture supernatant. After purification by Ni-NTA resin column and digestion by SUMO protease, 5.5 mg of plectasin with a purity of 94 % was obtained from 1 L fermentation culture. Recombinant plectasin was found inhibition activity against S. pneumoniae, S. aureus and S. epidermidis. These results indicate that the maltose-induced expression system may be a safe and efficient way for the large-scale production of soluble peptides in B. subtilis.

Antifungal Peptides as Therapeutic Agents

“…Bacillus subtilis has also been explored as host for AFPs production. This includes plectasin (Zhang et al, 2015), cathelicidin (Luan et al, 2014), and the hybrid cecropin Amelittin (Ji et al, 2017) (Table 4). B. subtilis is a well-studied species, is non-pathogenic, has been approved by the Food and Drug Administration as a Generally Regarded As Safe (GRAS) microorganism and does not exhibit codon bias.…”

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

“…The drawback of this system is that the expression yield is sometimes low as compared to E. coli. For example, the yield of plectasin when expressed in B. subtilis (Zhang et al 2015a) was three times lower than that in E. coli (Chen et al 2015).…”

“…Bacillus subtilis (B. subtilis) has also been used as an alternative prokaryotic expression system to E. coli to produce AMPs including plectasin (Zhang et al 2015a), cathelicidin (Luan et al 2014), and hybrid cecropin A-melittin (Ji et al 2017). B. subtilis has rapid growth rate and facilitates simpler protein purification as it secretes recombinant proteins directly into culture media, thus, avoiding cell-disruption step.…”

“…Purification of AMPs is rationally designed based on the characteristics of different tags and AMPs in the post-cleavage mixture. Immobilized metal-affinity chromatography is most commonly used technique to bind His-tagged fusion proteins after the cleavage step, and the AMPs can be recovered from the flow-through fraction (Luan et al 2014;Wei et al 2018;Zhang et al 2014;Zhang et al 2015a). Size exclusion chromatography (SEC) separates the cleaved AMP from fusion tags based on the size difference in which the size gap should be large enough to allow better resolution (Sun et al 2018a;Tao et al 2014;Yi et al 2015).…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…Similar fusion partners such as SUMO [258a,260a] are also used in this scenario, followed by enzymatic cleavage to release the native, active peptides. Other expression hosts used in peptide production include P. pastoris, which may be useful in producing cysteinerich AMPs [263] that fold poorly in E. coli, [264] B. subtilis to express an anti-Gram positive fungal plectasin fused to SUMO, [265] and rice leaves. [266] The main alternative to recombinant peptide expression is solid phase peptide synthesis (SPPS), which is now largely dominated by the fluorenylmethyloxycarbonyl (Fmoc) approach.…”