NmLgtC | Biochem 3

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