3 Methods
3.1 OAg Extraction
and Purification
OAg extraction is performed directly on intact bacteria avoiding the
step of LPS extraction, hydrolyzing the OAg from the lipid A while
still attached to the bacterial membrane. The O-antigen chain plus
the core is released in solution, while the lipid A remains attached to
the bacterial membrane and pelleted by centrifugation. Here we
report the extraction, purification (Scheme 1), and analytical characterization of S. paratyphi A OAg, as an example (Fig. 2). The
same procedure has been applied to OAg from S. typhimurium,
S. enteritidis, and Shigella flexneri strains and can be applied with
modifications, for what concerns the purification steps, to OAg
from other strains.
1. Add acetic acid to the bacterial culture at a final concentration
of 1–2% (v/v), check the pH is in the range of 3.5–4.7, and
incubate at 100
C for 6 h (see Note 5).
2. At the end of hydrolysis, add 28% NH 4 OH, under chemical
hood or directly in the bioreactor, based on the scale used, to
increase the pH to around 6 and collect the supernatant by
centrifugation. Indicate this solution as “post-hydrolysis
supernatant.”
3. Concentrate the “post hydrolysis supernatant” (see Note 6)
five- to tenfold by Tangential Flow Filtration (TFF) with a
30-kDa molecular weight cut-off (MWCO), 200-cm
2 Hydrosart membrane. After concentration, perform diafiltration
against 20 diavolumes of 1 M NaCl and another 10 diavolumes
of water (see Note 7). Collect the retentate and indicate this
solution as “TFF-1 retentate.”
l
Remark: membrane with a different molecular weight
cut-off could be used according to the OAg size.
4. Add citrate buffer 200 mM pH 2.7 with continuous stirring to
the “TFF-1 retentate” to have a final citrate concentration of
20 mM. Check that pH of the OAg solution is around 3. Mix at
RT for 30 Æ 5 min. After this time, collect the supernatant after
centrifugation (12,000 RCF at 15
C for 30 min) (see Note 8).
Indicate this solution as “post-pH 3” (see Note 9).
5. To “post-pH 3” (see Note 10) add, while stirring at RT,
500 mM Na 2 HPO 4 , absolute ethanol (EtOH), and 5 M
CaCl 2 to give 18 mM NaH 2 PO 4 , 24% EtOH, (v/v), and
200 mM CaCl 2 concentrations in the final mixture. Perform
this addition within a chemical hood. Mix at room temperature
for 30 Æ 5 min. After this time, collect the supernatant after
centrifugation (12,000 RCF at 15
C for 30 min) (see Note
11). Indicate this solution as “post-EtOH/CaCl 2 .”
276
Francesca Micoli et al.
3.1 OAg Extraction
and Purification
OAg extraction is performed directly on intact bacteria avoiding the
step of LPS extraction, hydrolyzing the OAg from the lipid A while
still attached to the bacterial membrane. The O-antigen chain plus
the core is released in solution, while the lipid A remains attached to
the bacterial membrane and pelleted by centrifugation. Here we
report the extraction, purification (Scheme 1), and analytical characterization of S. paratyphi A OAg, as an example (Fig. 2). The
same procedure has been applied to OAg from S. typhimurium,
S. enteritidis, and Shigella flexneri strains and can be applied with
modifications, for what concerns the purification steps, to OAg
from other strains.
1. Add acetic acid to the bacterial culture at a final concentration
of 1–2% (v/v), check the pH is in the range of 3.5–4.7, and
incubate at 100
C for 6 h (see Note 5).
2. At the end of hydrolysis, add 28% NH 4 OH, under chemical
hood or directly in the bioreactor, based on the scale used, to
increase the pH to around 6 and collect the supernatant by
centrifugation. Indicate this solution as “post-hydrolysis
supernatant.”
3. Concentrate the “post hydrolysis supernatant” (see Note 6)
five- to tenfold by Tangential Flow Filtration (TFF) with a
30-kDa molecular weight cut-off (MWCO), 200-cm
2 Hydrosart membrane. After concentration, perform diafiltration
against 20 diavolumes of 1 M NaCl and another 10 diavolumes
of water (see Note 7). Collect the retentate and indicate this
solution as “TFF-1 retentate.”
l
Remark: membrane with a different molecular weight
cut-off could be used according to the OAg size.
4. Add citrate buffer 200 mM pH 2.7 with continuous stirring to
the “TFF-1 retentate” to have a final citrate concentration of
20 mM. Check that pH of the OAg solution is around 3. Mix at
RT for 30 Æ 5 min. After this time, collect the supernatant after
centrifugation (12,000 RCF at 15
C for 30 min) (see Note 8).
Indicate this solution as “post-pH 3” (see Note 9).
5. To “post-pH 3” (see Note 10) add, while stirring at RT,
500 mM Na 2 HPO 4 , absolute ethanol (EtOH), and 5 M
CaCl 2 to give 18 mM NaH 2 PO 4 , 24% EtOH, (v/v), and
200 mM CaCl 2 concentrations in the final mixture. Perform
this addition within a chemical hood. Mix at room temperature
for 30 Æ 5 min. After this time, collect the supernatant after
centrifugation (12,000 RCF at 15
C for 30 min) (see Note
11). Indicate this solution as “post-EtOH/CaCl 2 .”
276
Francesca Micoli et al.