products can be found elsewhere [20, 23]). The active site of a
GT-A fold contains a conserved motif (DXD) that interacts
primarily with the phosphate groups of the nucleotide donor
through the coordination of a divalent cation [25]. Thus, divalent cations are usually added to the reaction buffer. We
observed sufficient Cps4B activity in the presence of 10 mM
MgCl 2 . However, other cations (e.g., Mn
2+ , Co
2+
, Fe
2+ , Cu
2+
,
and Zn
2+
) have been reported as essential cofactors for enzyme
activity of other GT-A folded or non-GT-A folded proteins
[10, 25, 26] and should be considered as additives in the
reaction.
5. It is of note that Cps4B and Cps11D synthesize the polymer de
novo in the absence of an acceptor substrate. However, many
polymerases need a priming acceptor (such as hydrolyzed polymer fragments) to initiate polymer synthesis [1].
6. The signals obtained in the HPLC-AEC assay result from the
absorption of light by the nucleobases (absorption maximum
approx. 260 nm) that are part of the nucleotides (e.g., CMP,
UMP, UDP) and nucleotide activated substrates (e.g.,
UDP-Gal, CDP-Gro). The signals obtained for (UV-active)
polymers result from the absorption of light by the N-acetyl
groups at 214 nm, which are present in the polymer generated
by Cps4B and lacking in the polymer generated by Cps11D (see
Fig. 2).
7. CarboPac™ columns are intended to be used for the separation
of charged and noncharged carbohydrates under high pH conditions. However, the separation of negatively charged molecules under neutral pH conditions is possible as well.
8. The filtered and autoclaved buffer can be used for several
measurements. Please check the clarity and purity before
using the buffer in the HPLC assay.
9. Nucleotides and nucleotide sugars are detected at 280 nm,
which deviates from the wavelength of maximum absorption
(approx. 260 nm for most nucleotides), leading to a lower
UV-signal intensity and thus allowing a direct injection of
sample into the HPLC system without prior dilution of the
reaction mixture. If nucleotide concentrations far below 1 mM
are required in the enzymatic reaction, changing the detection
wavelength to 260 nm should be considered to increase the
signal intensity.
10. Since fragments of group 2 polymers have the same charge-tomass ratio, separation occurs on the basis of fragment size
[24]. Consequently, improved resolution can be achieved by
increasing the gel length. The gel shown in Fig. 7 measures
$13.5 cm (height) Â ~11 cm (width). In addition, decreasing
the sample load also contributes to better resolution of single
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
325
GT-A fold contains a conserved motif (DXD) that interacts
primarily with the phosphate groups of the nucleotide donor
through the coordination of a divalent cation [25]. Thus, divalent cations are usually added to the reaction buffer. We
observed sufficient Cps4B activity in the presence of 10 mM
MgCl 2 . However, other cations (e.g., Mn
2+ , Co
2+
, Fe
2+ , Cu
2+
,
and Zn
2+
) have been reported as essential cofactors for enzyme
activity of other GT-A folded or non-GT-A folded proteins
[10, 25, 26] and should be considered as additives in the
reaction.
5. It is of note that Cps4B and Cps11D synthesize the polymer de
novo in the absence of an acceptor substrate. However, many
polymerases need a priming acceptor (such as hydrolyzed polymer fragments) to initiate polymer synthesis [1].
6. The signals obtained in the HPLC-AEC assay result from the
absorption of light by the nucleobases (absorption maximum
approx. 260 nm) that are part of the nucleotides (e.g., CMP,
UMP, UDP) and nucleotide activated substrates (e.g.,
UDP-Gal, CDP-Gro). The signals obtained for (UV-active)
polymers result from the absorption of light by the N-acetyl
groups at 214 nm, which are present in the polymer generated
by Cps4B and lacking in the polymer generated by Cps11D (see
Fig. 2).
7. CarboPac™ columns are intended to be used for the separation
of charged and noncharged carbohydrates under high pH conditions. However, the separation of negatively charged molecules under neutral pH conditions is possible as well.
8. The filtered and autoclaved buffer can be used for several
measurements. Please check the clarity and purity before
using the buffer in the HPLC assay.
9. Nucleotides and nucleotide sugars are detected at 280 nm,
which deviates from the wavelength of maximum absorption
(approx. 260 nm for most nucleotides), leading to a lower
UV-signal intensity and thus allowing a direct injection of
sample into the HPLC system without prior dilution of the
reaction mixture. If nucleotide concentrations far below 1 mM
are required in the enzymatic reaction, changing the detection
wavelength to 260 nm should be considered to increase the
signal intensity.
10. Since fragments of group 2 polymers have the same charge-tomass ratio, separation occurs on the basis of fragment size
[24]. Consequently, improved resolution can be achieved by
increasing the gel length. The gel shown in Fig. 7 measures
$13.5 cm (height) Â ~11 cm (width). In addition, decreasing
the sample load also contributes to better resolution of single
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
325