Expression of Low Endotoxin 3-O-Sulfotransferase in Bacillus subtilis and Bacillus megaterium
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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 104–105-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources.
Keywords3-O-Sulfotransferase Bioengineered heparin Endotoxin free Bacillus subtilis Bacillus megaterium Nonanimal sources
This work was supported by grants funded by the National Institutes of Health HL101721 and HL096972 (RJL), the Bioengineered Heparin Consortium, and 863 Hi-Tech Research and Development Program of the People’s Republic of China (project no. 2012AA022300). The authors would also like to thank Dr. Sui-Lam Wong (University of Calgary), Dr. Xiaozhou Zhang (Virginia Tech), and Dr. Cynthia Collins (Rensselaer Polytechnic Institute) for supplying bacterial strains and plasmids.
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