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    High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin

    Author
    Zhang, J.; Suflita, M.; Fiaschetti, C.M.; Li, G.; Li, L.; Zhang, F.; Dordick, J.S.; Linhardt, Robert J.
    ORCID
    https://orcid.org/0000-0003-2219-5833
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    HIGH CELL DENSITY CULTIVATION OF A RECOMBINANT ESCHERICHIA COLI.pdf (727.8Kb)
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    Date Issued
    2015-01-01
    Subject
    Biology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
    Degree
    Terms of Use
    In Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/;
    Full Citation
    High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin, J. Zhang, M. Suflita, C.M. Fiaschetti, G. Li, L. Li, F. Zhang, J.S. Dordick, R. J. Linhardt, Journal of Applied Microbiology, 118, 92-98, 2014.
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    URI
    https://hdl.handle.net/20.500.13015/5316; https://doi.org/10.1111/jam.12684
    Abstract
    Aims: One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. Methods and results: The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl β-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)).Conclusions: The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated.Significance and impact of the study: The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market.;
    Description
    Journal of Applied Microbiology, 118, 92-98; Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
    Department
    The Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
    Publisher
    Wiley
    Relationships
    The Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; Journal of Applied Microbiology; https://harc.rpi.edu/;
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    A full text version is available in DSpace@RPI;
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