title: An enhanced protocol for expression and purification of heat-stable enterotoxin of enterotoxigenic Escherichia coli.
abstract: We present an improved protocol for expression and purification of heat-stable enterotoxin (STa) of enterotoxigenic Escherichia coli (ETEC). In this protocol, controlled growth conditions at different pHs (7.4, 8.0, and 8.6) were adopted using a bioreactor. In addition, specific adsorbent resins, methacrylate, were used for STa purification. The bioreactor provided optimal ETEC growth at pH 7.4 with high STa production. Furthermore, methacrylate bounded specifically to STa and dramatically enhanced the purification process of STa. The STa-specific activity was high (8.9 × 10(6) units/mg protein), and the minimal effective dose of STa required for production of gut weight to remaining body weight ratio ≥ 0.083 was recorded as less than 0.2 ng in 2-3 days old suckling mice. The protocol presented, produces highly purified STa as documented by matrix-assisted laser desorption ionization-time of flight mass spectroscopy/. Also, as compared with the traditional methods, this procedure is trouble-free and practical for scale-up production and purification of STa peptides.
authors.affiliation: Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48823, USA. aref71@hotmail.com
authors.forename: Nasr-Eldin M
authors.initials: NE
authors.lastname: Aref
authors.forename: A Mahdi
authors.initials: AM
authors.lastname: Saeed
mesh_terms: D000327:Adsorption; D000818:Animals; D019149:Bioreactors; D056148:Chromatography, Reverse-Phase; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D041981:Gastrointestinal Tract / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000737:chemistry; D051379:Mice; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004768:Enterotoxins; D008689:Methacrylates