Molecular chaperones (TrxA, SUMO, Intein, and GST) mediating expression, purification, and antimicrobial activity assays of plectasin in Escherichia coli

Abstract

Plectasin (PS) is the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella, and active against Streptococcus pneumoniae and S. aureus, including antibiotic-resistant pathogens. To establish a bacterium-based production system, we compared the efficiency of four molecular chaperones and corresponding cleavage to the expression and purification of plectasin. The results showed that the yield of plectasin combined with thioredoxin A (TrxA) and small ubiquitin-related modifier (SUMO) was at a higher level (0.0356 and 0.0358 g L(-1), respectively) than that with intein (0.0238 g L(-1)) and glutathione-S-transferase (GST) (0.0243 g L(-1)). TrxA-plectasin, SUMO-plectasin, and 2-plectasin were cleaved at the correct site and purified, but their considerable amount was not cleaved and remained as a fusion peptide. The antimicrobial activity of plectasin cleaved from SUMO--plectasin against methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant S. pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE)--was stronger than ampicillin (Amp) for the same amount of substance (P ≤ 0.05). This is the first study to complete and compare the effect of different molecular chaperones and corresponding cleavage with the expression and purification of plectasin in the Escherichia coli expression system, which laid the foundation for future research and may develop the application and production of plectasin.

Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700

“…SUMO can dramatically enhance expression and chaperone correct protein folding in the soluble form. This fusion technology has recently been used to express several AMPs, including scolopin 118, CM419 and plectasin20, in E. coli expression system. However, the purification procedure, which includes breaking host cells to release the fusion proteins and supplementing additional SUMO protease to liberate the protein of interest from SUMO, somewhat inhibits the application of this technology.…”

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

“…In previous study, Chen et al expressed plectasin in E.coli using SUMO technology and obtained 35.8 mg/L fusion protein, which is lower than the fusion protein we obtained [2]. Zhang et al and Yang et al have obtained 3.5 mg and 1.75 mg of recombinant plectasin polypeptide, respectively, from 1 L of E. coli fermentation culture medium, which is lower than the 5.5 mg/L of recombinant plectasin polypeptide expressed by B. subtilis in this study [10,24].…”

Fungal proteinaceous compounds with multiple biological activities

“…The yields of plectasin combined with s m a l l u b i q u i t i n -r e l a t e d m o d i f i e r (S U M O ) an d thioredoxin A were higher than those obtained by combination with glutathione-S-transferase and intein. The inhibitory effect of plectasin cleaved from SUMOplectasin against MRSA, vancomycin-resistant enterococci and penicillin-resistant S. pneumoniae was more striking than that elicited by the same amount of ampicillin (Chen et al 2015a). Recombinant and synthetic plectasin demonstrated potent thermostable and pHstable antibacterial activity against gram-positive bacteria, including antibiotic-resistant bacterial species, in particular penicillin-resistant Enterococcus faecium (Chen et al 2015b).…”