Alkaline phosphatase e coli c75

The reaction was performed at 37 °C in a reaction mixture (200 μL) containing 100 mM 3-(N-morpholino)propanesulfonic acid (pH 7.3), 10 mM MnCl₂, 350 μM MUC1 T-Ser₁₉, 1 mM CMP-Neu5Ac, 1 mM phenylmethylsulfonyl fluoride, complete protease inhibitor (Roche Diagnostics), alkaline phosphatase E. coli C75 (2 U, Takara Bio) and 23 μU ST6GalNAc1. After 24 hours of incubation, ST6GalNAc1 (15 μU, 70 μL) and CMP-Neu5Ac (100 nmol, 1 μL) were added to the reaction mixture. After further 24 hours of incubation at 37 °C, the reaction mixture was heated at 95 °C for five minutes and was lyophilised. The resulting residue was dissolved with 150 μL of water and filtered over a 0.22 μm filter. The filtrate was subjected to HPLC in the same condition with analytical HPLC as described above. The α2,6-sialylated product was isolated and dried by lyophilisation. The residue was dissolved with 50 μL of water, and 6 μL of Glycobuffer 4 (10×, New England BioLabs) was added to the mixture, followed by 15 μL of β1-3,4 galactosidase (120 U, from bovine testis, New England BioLabs). The reaction mixture was incubated for 18 hours at 37 °C, filtered over a 0.22 μm filter, and subjected to HPLC purification as same as after sialylation. The purified fraction was lyophilised and gave MUC1 STn-Ser₁₉ (16.3 nmol, 23% yield). MALDI-TOF MS: C₁₂₈H₂₀₁N₃₆O₄₈ [M + H]⁺ calculated (m/z) 3010.44, found (m/z) 3010.42.

Plasmid purification kits (QIAGEN Plasmid Maxi Kit, QIAprep Spin Miniprep Kit) were purchased from QIAGEN (Venlo, The Netherlands). Restriction enzyme, T4 polynucleotide kinase, alkaline phosphatase (E. coli C75), DNA ligation kit (DNA Ligation Kit Ver.1), DNA polymerase (TAKARA Premix Taq, EX Taq version), and Site-Directed Mutagenesis kit (Mutan^®- Super Express Km) were purchased from Takara Bio (Kusatsu, Japan). Heparin was purchased from Mochida Pharmaceuticals (Shinjuku City, Tokyo) and Block Ace from Sumitomo Dainippon Pharma (Osaka, Japan). Blue Sepharose 6-Fast Flow, 5 mL HiTrap Phenyl HP, and 5 mL HiTrap Q XL were purchased from GE Healthcare Japan (Tokyo, Japan). HE staining reagents were purchased from Muto Chemical (Tokyo, Japan). Mouse anti-α-SMA antibody (cat#:ab5694) was purchased from abcam (Cambridge, UK). All other reagents and solvents were commercially available special grade products, and the water used as the solvent was ion-exchanged water or Milli-Q water.