12. Both acetic acid and alcian blue (a cationic dye for the staining
of acidic polysaccharides such as polysialic acid, glycosaminoglycans, and phosphate-containing polymers) fixate the polymers in the gel and prevent smaller species from diffusing from
the gel [24, 27]. In our experience, oligosaccharides !4 monomeric repeating units can be visualized using this protocol. It is
recommended to use alcian blue in acetic acid rather than in
water, because acetic acid enhances the solubility of the dye,
prevents precipitation and thus allows the reuse of the staining
solution [24]. If the alcian blue solution is used without
subsequent silver staining, only large polymer fragments are
visualized.
13. The periodic acid oxidizes 1,2-diol groups of monosaccharide
subunits of the polymers to aldehydes that can be revealed by
silver staining performed in the following step [27].
14. Positively charged silver ions bind to negative groups in the
polymer (e.g., phosphate groups, carboxyl groups). Formaldehyde reduces the silver ions to silver, leading to the brown/
black colour.
15. The addition of acetic acid changes the pH which stops the
staining process.
16. The HPLC method for nucleotide separation allows the analysis of substrate turnover. If the polymerase precipitates before
all substrates are used up, remove precipitates by centrifugation
(20,000 Â g) and add additional polymerase to the reaction
until substrate consumption is completed and synthesis yield is
maximized. In the long run, buffer conditions should be optimized for each individual polymerase and might deviate from
the conditions used in this protocol for Cps4B and Cps11D.
17. Some nucleotide activated substrates are less stable under certain conditions. In our experience, UDP-GlcNAc is stable in
the above-listed reaction buffer overnight at 37
C, whereas,
for example, UDP-Gal quickly breaks down to UMP and
Gal-1P [21], especially when magnesium ions are replaced by
manganese ions. To minimize unwanted hydrolysis, reaction
time can be shortened or the enzyme concentration can be
increased to maximize substrate uptake.
18. The temperature optimum for most polymerases is 37
C.
However, many polymerases are also active at room temperature. In some cases (e.g., upscaled reactions [15], solid-phase
coupling of enzymes [12]), decreasing the reaction temperature might facilitate the experimental setup.
19. If necessary, minimize the duration and temperature of the
heat shock since temperature-sensitive substrates or polymers
might degrade.
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
327
of acidic polysaccharides such as polysialic acid, glycosaminoglycans, and phosphate-containing polymers) fixate the polymers in the gel and prevent smaller species from diffusing from
the gel [24, 27]. In our experience, oligosaccharides !4 monomeric repeating units can be visualized using this protocol. It is
recommended to use alcian blue in acetic acid rather than in
water, because acetic acid enhances the solubility of the dye,
prevents precipitation and thus allows the reuse of the staining
solution [24]. If the alcian blue solution is used without
subsequent silver staining, only large polymer fragments are
visualized.
13. The periodic acid oxidizes 1,2-diol groups of monosaccharide
subunits of the polymers to aldehydes that can be revealed by
silver staining performed in the following step [27].
14. Positively charged silver ions bind to negative groups in the
polymer (e.g., phosphate groups, carboxyl groups). Formaldehyde reduces the silver ions to silver, leading to the brown/
black colour.
15. The addition of acetic acid changes the pH which stops the
staining process.
16. The HPLC method for nucleotide separation allows the analysis of substrate turnover. If the polymerase precipitates before
all substrates are used up, remove precipitates by centrifugation
(20,000 Â g) and add additional polymerase to the reaction
until substrate consumption is completed and synthesis yield is
maximized. In the long run, buffer conditions should be optimized for each individual polymerase and might deviate from
the conditions used in this protocol for Cps4B and Cps11D.
17. Some nucleotide activated substrates are less stable under certain conditions. In our experience, UDP-GlcNAc is stable in
the above-listed reaction buffer overnight at 37
C, whereas,
for example, UDP-Gal quickly breaks down to UMP and
Gal-1P [21], especially when magnesium ions are replaced by
manganese ions. To minimize unwanted hydrolysis, reaction
time can be shortened or the enzyme concentration can be
increased to maximize substrate uptake.
18. The temperature optimum for most polymerases is 37
C.
However, many polymerases are also active at room temperature. In some cases (e.g., upscaled reactions [15], solid-phase
coupling of enzymes [12]), decreasing the reaction temperature might facilitate the experimental setup.
19. If necessary, minimize the duration and temperature of the
heat shock since temperature-sensitive substrates or polymers
might degrade.
Enzymatic Polymer Synthesis for Glycoconjugate Vaccines
327