Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium Wenya Wang & Jacob A. Englaender & Peng Xu & Krunal K. Mehta & Jiraporn Suwan & Jonathan S. Dordick & Fuming Zhang & Qipeng Yuan & Robert J. Linhardt & Mattheos Koffas Received: 16 March 2013 / Accepted: 24 July 2013 / Published online: 4 August 2013 # Springer Science+Business Media New York 2013 Abstract A key enzyme for the biosynthesis and bioengineering of heparin, 3- O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were Appl Biochem Biotechnol (2013) 171:954–962 DOI 10.1007/s12010-013-0415-8 Wenya Wang and Jacob A. Englaender equally contributed to this work. W. Wang : Q. Yuan College of Life Science and Technology, Beijing University of Chemical Technology, Box 75, Beijing 100029, China W. Wang : J. Suwan : R. J. Linhardt Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA P. Xu : K. K. Mehta : J. S. Dordick : F. Zhang : R. J. Linhardt : M. Koffas Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA J. A. Englaender : J. S. Dordick : R. J. Linhardt Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA J. S. Dordick : R. J. Linhardt Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA J. S. Dordick Department of Materials Science and Engineering Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA R. J. Linhardt (*) : M. Koffas (*) Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Biotech 4005, 110 8th Street, Troy, NY 12180, USA e-mail: linhar@rpi.edu e-mail: koffam@rpi.edu