title: Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli.
abstract: Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside.
authors.affiliation: Institut für Organische Chemie, Julius-Maximilians-Universität, Am Hubland, 97074, Würzburg, Germany.
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Ortiz-Soto
authors.affiliation: Institut für Organische Chemie, Julius-Maximilians-Universität, Am Hubland, 97074, Würzburg, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Seibel
mesh_terms: D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005960:Glucosides / Q000096:biosynthesis; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; D050862:Oxidoreductases Acting on Sulfur Group Donors; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D015703:Antigens, CD; D005960:Glucosides; D058619:Maltose-Binding Proteins; D018832:Molecular Chaperones; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011993:Recombinant Fusion Proteins; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase; D050862:Oxidoreductases Acting on Sulfur Group Donors; C093493:ST6GAL1 protein, human; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase