title: 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.
authors.affiliation: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Chen
authors.affiliation: Nanfang Hospital, First Clinical Medical College, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Shi
authors.affiliation: Department of Respiratory Diseases, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, People's Republic of China.
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.affiliation: Nanfang Hospital, First Clinical Medical College, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Yaoan
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Biosafety Level-3 Laboratory, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Department of Respiratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Songwen
authors.initials: S
authors.lastname: Guo
authors.affiliation: Biosafety Level-3 Laboratory, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, People's Republic of China.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000378:metabolism; D047668:Inteins; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D055550:Protein Stability; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D025842:SUMO-1 Protein / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D018832:Molecular Chaperones; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C504857:plectasin; D013879:Thioredoxins; D005982:Glutathione Transferase
keywords: cleavage; expression; molecular chaperone; plectasin; purification