mix 1 mL of 1 M Tris–HCL (pH 8.0) with 9 mL of water.
Gently mix.
4. 25 mM dithiothreitol (DTT) (see Note 2). For 10 mL of a 40Â
stock (1 M DTT) dissolve 1.54 g of DTT in 10 mL of water.
Prepare 0.5 mL to 1 mL aliquots for storage at À20
C. Dilute
25 μL of 1 M DTT with 975 μL of water to obtain a 25 mM
DTT working solution. Gently mix. Use the freshly prepared
DTT for the preparation of the assay buffer. Use it only once
and discard the solution after usage.
5. 100 mM nucleotide activated substrates. Weigh an appropriate
amount of nucleotide activated substrate and dissolve in an
appropriate amount of water to obtain a 100 mM stock solution (see Note 3). Prepare aliquots for the storage at À80
C.
UDP-Glc/UDP-GalNAc and UDP-Gal/CDP-Gro are substrates of Cps4B and Cps11D, respectively (see Fig. 2).
6. 50 mM magnesium chloride (MgCl 2 ) (see Notes 4 and 5). For
100 mL of a 20Â stock solution (1 M MgCl 2 ) weigh 20.3 g of
MgCl 2 (*6H 2 O) in a 100 mL volumetric flask and add water to
the graduation mark. Dissolve the salt, sterile-filter (22 μm) the
buffer and transfer it to a 100 mL flask for autoclavation. Dilute
50 μL of the 1 M MgCl 2 stock solution with 950 μL of water
for the preparation of 1Â MgCl 2 (50 mM).
2.2 HPLC Assay
Prepare all solutions with ultrapure water and analytical grade
reagents. The method requires an HPLC system equipped with a
UV detector allowing wavelengths of 280 nm and 214 nm and
corresponding software for data analysis. The column oven needs
to be equipped with a column suitable for anion exchange chromatography. Results were obtained with a Prominence UFLC-XR
(Shimadzu) equipped with a SPD-20AV detector (Shimadzu) (see
Note 6) and a CarboPac™ PA-100 column 2 Â 250 mm + guard
column (Dionex™) (see Note 7).
1. 1 M Tris–HCl (pH 8.0). Weigh 121.14 g of Tris in a 1 L
volumetric flask and add 600 mL of water. After the salt is
completely dissolved, adjust the pH to 8.0 with HCl. Add
water to a volume of 1 L. Filter the buffer and transfer it to a
1 L flask for autoclavation.
2. 5 M NaCl. Weigh 292.2 g of NaCl in a 1 L volumetric flask and
add ~950 mL of water. Dissolve the salt using a magnetic
stirrer. Add water to a volume of 1 L after NaCl is completely
dissolved. Filter the buffer and transfer it to a flask for
autoclavation.
3. Preparation of mobile phase 1: 20 mM Tris–HCl (pH 8.0).
Transfer 20 mL of the 1 M Tris–HCl buffer to a 1 L volumetric
flask and add water to the graduation mark. Filter the buffer
and transfer it to a flask for autoclavation (see Note 8).
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
317
Gently mix.
4. 25 mM dithiothreitol (DTT) (see Note 2). For 10 mL of a 40Â
stock (1 M DTT) dissolve 1.54 g of DTT in 10 mL of water.
Prepare 0.5 mL to 1 mL aliquots for storage at À20
C. Dilute
25 μL of 1 M DTT with 975 μL of water to obtain a 25 mM
DTT working solution. Gently mix. Use the freshly prepared
DTT for the preparation of the assay buffer. Use it only once
and discard the solution after usage.
5. 100 mM nucleotide activated substrates. Weigh an appropriate
amount of nucleotide activated substrate and dissolve in an
appropriate amount of water to obtain a 100 mM stock solution (see Note 3). Prepare aliquots for the storage at À80
C.
UDP-Glc/UDP-GalNAc and UDP-Gal/CDP-Gro are substrates of Cps4B and Cps11D, respectively (see Fig. 2).
6. 50 mM magnesium chloride (MgCl 2 ) (see Notes 4 and 5). For
100 mL of a 20Â stock solution (1 M MgCl 2 ) weigh 20.3 g of
MgCl 2 (*6H 2 O) in a 100 mL volumetric flask and add water to
the graduation mark. Dissolve the salt, sterile-filter (22 μm) the
buffer and transfer it to a 100 mL flask for autoclavation. Dilute
50 μL of the 1 M MgCl 2 stock solution with 950 μL of water
for the preparation of 1Â MgCl 2 (50 mM).
2.2 HPLC Assay
Prepare all solutions with ultrapure water and analytical grade
reagents. The method requires an HPLC system equipped with a
UV detector allowing wavelengths of 280 nm and 214 nm and
corresponding software for data analysis. The column oven needs
to be equipped with a column suitable for anion exchange chromatography. Results were obtained with a Prominence UFLC-XR
(Shimadzu) equipped with a SPD-20AV detector (Shimadzu) (see
Note 6) and a CarboPac™ PA-100 column 2 Â 250 mm + guard
column (Dionex™) (see Note 7).
1. 1 M Tris–HCl (pH 8.0). Weigh 121.14 g of Tris in a 1 L
volumetric flask and add 600 mL of water. After the salt is
completely dissolved, adjust the pH to 8.0 with HCl. Add
water to a volume of 1 L. Filter the buffer and transfer it to a
1 L flask for autoclavation.
2. 5 M NaCl. Weigh 292.2 g of NaCl in a 1 L volumetric flask and
add ~950 mL of water. Dissolve the salt using a magnetic
stirrer. Add water to a volume of 1 L after NaCl is completely
dissolved. Filter the buffer and transfer it to a flask for
autoclavation.
3. Preparation of mobile phase 1: 20 mM Tris–HCl (pH 8.0).
Transfer 20 mL of the 1 M Tris–HCl buffer to a 1 L volumetric
flask and add water to the graduation mark. Filter the buffer
and transfer it to a flask for autoclavation (see Note 8).
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
317