Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Figure Lengend Snippet: Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
Article Snippet: The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem.
Techniques: Expressing, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation