A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A |
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Authors: | Giuseppina Pieretti Manuela Cipolletti Daniele D’Alonzo Alberto Alfano Donatella Cimini Marcella Cammarota Giovanni Palumbo Mariateresa Giuliano Mario De Rosa Chiara Schiraldi Michelangelo Parrilli Emiliano Bedini Maria Michela Corsaro |
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Affiliation: | 1. Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, via Cintia 4, 80126, Napoli, Italy 2. Department of Experimental Medicine, Second University of Naples, via de Crecchio 7, 80138, Napoli, Italy
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Abstract: | Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates. |
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