Chromobacterium violaceum b1-3 galactosyltransferase

Abbreviation 

Cvb3GalT

Source

Chromobacterium violaceum

Activity

Transfer Gal from UDP-Gal to GlcNAc via a b1,3-linkage

Expression

&

Purification

Expression system:    pET22b with C-terminal His6-tag, E. coli BL21(DE3)

Induction condition:  1 mM IPTG, 20 °C, 20 h, 200 rpm, Luria LB broth

Purification

1) Suspend cells collected from 1 L medium in ice-cold 40 mL Buffer-A + 10% glycerol

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, 50 % Glycerol

Optimal Condition

37 °C

6.5 - 9.5

MnCl2, MgCl2

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

GlcNAcb1-3Lac

Donor:

Acceptor:

Kinetics:

Substrate Specificity

  • McArthur J.B., Yu H., and Chen X. A Bacterial b1–3-Galactosyltransferase Enables Multigram-Scale Synthesis of Human Milk Lacto-N-tetraose (LNT) and Its Fucosides. ACS Cata, 2019, 9, 10721.

References