title: Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli.
abstract: The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.
authors.affiliation: Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, People's Republic of China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhao
authors.forename: Lin
authors.initials: L
authors.lastname: Kang
authors.forename: Shan
authors.initials: S
authors.lastname: Gao
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Libo
authors.initials: L
authors.lastname: Su
authors.forename: Wenwen
authors.initials: W
authors.lastname: Xin
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Su
authors.forename: Jinglin
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001681:Biological Assay; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000737:chemistry; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes; D004926:Escherichia coli; D005822:Genetic Vectors / Q000378:metabolism; D006461:Hemolysis; D006639:Histidine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013879:Thioredoxins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens