Protein Expression and PuriWcation 38 (2004) 170–176 www.elsevier.com/locate/yprep 1046-5928/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2004.08.011 A method for the puriWcation of Shiga-like toxin 1 subunit B using a commercially available galabiose–agarose resin  Maria Teresa Tarragó-Trani a , Brian Storrie b,¤ a Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA b Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Received 18 May 2004, and in revised form 10 August 2004 Available online 5 October 2004 Abstract We describe a procedure for the aYnity puriWcation of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose–agarose resin. Recombinant SLTB was puriWed to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio chol- erae 0395 N1/pSBC54. SDS–PAGE of the aYnity puriWed SLTB showed one band of 8 kDa MW. SLTB puriWed by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose–agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose–agarose resin could isolate roughly 1 mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost.  2004 Elsevier Inc. All rights reserved. Keywords: Shiga-like toxin subunit B; Galabiose; AYnity chromatography Shiga-like toxins (1 and 2) (SLT) 1 are toxins produced by some Escherichia coli strains that are structurally and biologically related to Shiga toxin (ST) produced by Shi- gella dysenteriae [1]. These toxins cause severe illnesses such as hemolytic colitis and hemolytic uremic syn- drome. The protein structure of Shiga and Shiga-like toxins consists of two subunits, termed A and B. Subunit A is an N-glycosidase that cleaves a unique adenine resi- due in the 28S rRNA of the 60S ribosomal subunit caus- ing inactivation of protein synthesis. One subunit A associates with a pentamer of Wve identical B subunits to give the holotoxin. Subunit B (SLTB) binds to the cell surface glycosphingolipid, globotriaosylceramide or Gb 3 (Gal1-4Gal1-4Glc-Cer), which promotes toxin inter- nalization by receptor-mediated endocytosis. The holo- toxin, once in endosomes, uses cellular retrograde transport mechanisms to move to the Golgi apparatus and endoplasmic reticulum (ER) [2]. In the ER, the sub- unit A is translocated to the cytosol where it inactivates protein synthesis, hence producing cell killing. Due to the fatality of ST/SLT, especially in young children, methods for purifying and detecting the toxin have been of importance for generation of reagents to neutralize the toxin (immunoglobulins, synthetic glyco- conjugates) [3,4]. In recent years, owing to the ability of ST/SLT to enter cells through receptor binding, the non- toxic B subunit (STB/SLTB) has been sought as a vehicle to deliver therapeutic agents to target cells. Some  Supported by grants from Carilion Biomedical Institute, Roa- noke, VA 24011, USA. Optical Sciences and Engineering Research Center, Virginia Tech, Blacksburg, VA 24061, USA; and the National Institutes of Health (GM65233), Bethesda, MD, USA. * Corresponding author. Fax: +1 501 686 8167. E-mail address: StorrieBrian@uams.edu (B. Storrie). 1 Abbreviations used: ST, Shiga toxin; SLT, Shiga-like toxin; SLTB, Shiga-like toxin subunit B; Gb 3 , globotriaosylceramide; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, phos- phate-buVered saline; LB media/agar, Luria Bertani media/agar; MEM, minimum essential medium; FCS, fetal calf serum; DMEM, Dulbecco’s modiWed Eagle’s medium.