Neisseria meningitidis a1-4 galactosyltransferase

Abbreviation 

NmLgtC

Source

Neisseria meningitidis

Activity

Transfer Gal from UDP-Gal to Lac/LacNAc via an a1,4-linkage

Expression

&

Purification

Expression system:    pET15b with N-terminal His6-Tag , E. coli BL21(DE3)

Induction condition:  0.5 mM IPTG, 30 °C, 10 h, 200 rpm, Luria LB broth

Purification: >50 mg/L culture

1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A

2) Disrupt cells by a CF1 cell disruptor (Constant Systems), 20 kPsi, 4 °C

3) Centrifuge at 12000 x g, 4 °C, 30 min

4) Load supernatant to a 10 mL gravity column filled with 2 mL Ni-NTA resin (pre-equilibrated)

5) Wash with 50 mL Buffer-A, then 50 mM Buffer-B, finally elute with 10 mL Buffer-C.

6) Concentrate into 2.5 mL and change buffer to Buffer D using a PD-10 column. 

7) Store at 4 °C for up to 6 months, store at -80 °C for up to 1 year (avoid repeated freeze)

Buffers:   A: 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole;

                B: A + 15 mM imidazole; C: A + 250 mM imidazole. 

                D: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 % Glycerol

Optimal Condition

37 °C

7.5 - 8.5

MnCl2

50 mM Tris-HCl (pH 8.0), 22 mM donor, 20 mM acceptor, 20 mM MnCl2, incubate at 37 °C for 1 h to overnight.

Temperature

pH

Metal ion:

Example condition:   

UDP-Gal

Lac/LacNAc, dose not tolerate LacNAc on complex structures, e.g., N-glycan and O-glycans, dose not tolerate Lac/LacNAc on surfaces:

5 U/mg protein

Donor:

Acceptor:

Activity:

Substrate Specificity

  • Ban L., Pettit N., et al. Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry. Nat Chem Biol, 2012, 8, 769.

  • Zhang J., Kowal P., et al.Efficient chemoenzymatic synthesis of globotriose and its
    derivatives with a recombinant a1-4 galactosyltransferase. Carbohydr Res, 2002, 337, 969.

  • Koizumi S., Endo T., et al, Large-scale production of UDP-galactose and globotriose by coupling metabolically engineered bacteria. Nat Biotech, 1998, 16, 847 (NgLgtC)

References