| Summary: | <p>Abstract</p> <p>Background</p> <p>The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the <it>in vivo </it>half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically <it>in vitro </it>but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.</p> <p>Results and conclusion</p> <p>In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in <it>Escherichia coli </it>cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.</p>
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