Expression of soluble, active eukaryotic glycosyltransferases in prokaryotic organisms
a technology of eukaryotic glycosyltransferase and soluble eukaryotic glycosyltransferase, which is applied in the direction of transferases, drug compositions, enzymology, etc., can solve the problems of reducing the expectation of expression and the yield of active eukaryotic glycosyltransferase protein can be very
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example 1
Expression of Glucosyltransferases from the O-Linked Oligosaccharide Pathway
[0220]General Procedures
[0221]Constructs were designed to express maltose binding protein (MBP) fusions to amino-terminal truncations of the glycosyltransferases. Constructs are designated with a Δ(number) referring to the position of the last amino acid removed (for amino-terminal truncations) or the first amino acid removed (for carboxyl-terminal truncations) from the corresponding full-length protein. The following constructs were used: human MBP-GalNAc-T2 (Δ51), human GalNAc-T2 (Δ51Δ445), Drosophila MBP-Core-1-Gal-T1 (Δ50), porcine MBP-ST3Gal-1 (Δ45), and porcine MBP-SBD-ST3Gal-1 (Δ45; SBD is the starch binding domain tag, inserted between the MBP and the catalytic domains), and human MBP-ST6GalNAc-1 (Δ35). Nucleic acids encoding the enzymes were typically cloned into the BamHI-XhoI or BamHI-EcoRI sites of pCWin2-MBP or a version of pCWin2 with a modified 5′ UTR. See, e.g., PCT / US05 / 00302, filed Jan. 6, ...
example 2
Expression of Eukaryotic Glycosyltransferases from the N-Linked Oligosaccharide Pathway
[0240]General Procedures
[0241]Constructs were designed to express maltose binding protein (MBP) fusions to amino-terminal truncations of the glycosyltransferases. Constructs are designated with a Δ(number) referring to the number of amino acids removed from the amino-terminus of the corresponding native protein. The following constructs were used: human MBP-GnT1 (Δ103), bovine MBP-GalT1 (Δ129), and rat MBP-ST3Gal3 (Δ72) and MBP-SBD-ST3Gal3 (Δ72; SBD is the starch binding domain tag, inserted between the MBP and the catalytic domain), and human MBP-ST6GalNAc-1 (Δ35). For GnT1 and GalT1, an alternate version of each enzyme bearing a single missense mutation was also tested. Nucleic acids encoding the enzymes were typically cloned into the BamHI-XhoI or BamHI-EcoRI sites of pCWin2-MBP. See, e.g., PCT / US05 / 00302, filed Jan. 6, 2005, which is herein incorporated by reference for all purposes. Cloning w...
example 3
Expression of Eukaryotic Glycosyltransferases in Pseudomonas
[0252]General Procedures
[0253]Activities of the following eukaryotic glycosyltransferases were tested in a Pseudomonas expression system: two N-terminal truncations of porcine ST3Gal-1, and chicken ST6GalNAc-1. Constructs, with (Δ number) referring to the number of amino acids removed from the amino-terminus of the corresponding native protein, were: porcine ST3Gal-1 (Δ45), porcine ST3Gal-1 (Δ231), and chicken ST6GalNAc-1 (Δ231). The glycosyltransferases were fused to a Pseudomonas secretion sequence wherein expression is targeted to the periplasm, and / or were expressed as unfused proteins targeted to the cytoplasm. Expression was driven by the IPTG inducible Ptac promoter. Cloning was performed using standard techniques (e.g. Current Protocols in Molecular Biology, Ausubel, F M, et al, eds. John Wiley & Sons, Inc. 1998).
[0254]Plasmids comprising nucleic acids that express the glycosyltransferases were transformed into the...
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