Neisseria meningitidis b1-4 N-acetyl-hexosaminyltransferase
Abbreviation
NmLgtA
Source
Neisseria meningitidis MC58
Activity
Transfer GlcNAc/GalNAc from sugar donor to Gal via an b1,4-linkage
Expression
&
Purification
Expression system: pMAL-c2X with MBP and His6-tag , E. coli BL21(DE3)
Induction condition: 0. 1 mM IPTG, 16 °C, 20 h, 200 rpm, Luria LB broth
Purification:
1) Suspend cells in ice-cold 40 mL Buffer-A + 20 glycerol + 0.1% Triton-X100
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 -20 °C for up to 6 months
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, 50 % Glycerol
Optimal Condition
37 °C
8.0
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-GlcNAc/GalNAc and derivative
Lac/LacNAc
Donor:
Acceptor:
Kinetics:
Substrate Specificity
-
Li Y., Xue M., et al. Donor substrate promiscuity of bacterial b1–3-Nacetyl glucosaminyltransferases and acceptor substrate flexibility of b1–4-galactosyltransferases. Bioorg Med Chem, 2016, 24, 1696.
-
Guan W., Ban L., Combining carbochips and mass spectrometry to study the donor specificity for theNeisseria meningitidisb1,3-N-acetylglucosaminyltransferase LgtA. Bioorg Med Chem Lett, 2011, 21, 5025.
References