11. Load the beads onto fritted filtration columns attached to a
vacuum manifold system.
12. Allow the supernatant to pass through the column under
gravity.
(a) Take a sample of the flow through (FT) for denaturing
SDS-PAGE evaluation.
13. Wash beads with 20 CV of wash buffer.
(b) Take a sample of the wash (W) for denaturing SDS-PAGE
evaluation.
14. Elute protein with 4 CV of elution buffer under slow gravity
flow (see Note 5).
(a) Take a sample of the eluate (E) from each strain for
denaturing SDS-PAGE evaluation.
15. Record the absorption spectrum from 200 to 350 nm and
determine the concentration of AP205 VLPs taking into
account the protein and nucleic acid concentration according
to the algorithm of Porterfield and Zlotnick [12].
16. Concentrate and buffer exchange the purified proteins into
freezing buffer using 100 kDa MWCO centrifugal filters for
an approximate final concentration of 1–2 mg/mL (see Note
6).
17. After protein recovery from concentrators, centrifuge samples
at 20,000 Â g for 15 min to remove aggregates. Discard pellet.
18. Purified protein is stable at 4
C for at least 6 months or at
À80
C for years.
3.4 Analysis
of Purified VLPs
1. AP205 VLPs can be evaluated for particle assembly by native
electrophoresis on a 0.6% TAE-agarose gel (see Note 7). Fully
assembled particles are observed as distinct bands which stain
for both nucleic acids and protein (Fig. 2a).
2. Particle purity and glycosylation status can be qualitatively
evaluated by SDS-PAGE with Coomassie staining (Fig. 2b)
(see Notes 8 and 9).
3. Intact protein mass spectrometry can be used to semiquantitatively evaluate glycosylation of the coat proteins (Fig. 3).
4. Transmission electron microscopy can be used to visualize the
particle structure (Fig. 4) (see Note 10).
4 Notes
1. In the A. pleuropneumoniae genome, ORFs encoding the NGT
and α6GlcT are arranged sequentially as part of a polycistronic
operon. Previous studies have demonstrated that it is possible
to amplify these two ORFs and express them in a bicistronic
arrangement under the control of a single promoter [4].
Biosynthesis of Glycoconjugate VLPs
211
vacuum manifold system.
12. Allow the supernatant to pass through the column under
gravity.
(a) Take a sample of the flow through (FT) for denaturing
SDS-PAGE evaluation.
13. Wash beads with 20 CV of wash buffer.
(b) Take a sample of the wash (W) for denaturing SDS-PAGE
evaluation.
14. Elute protein with 4 CV of elution buffer under slow gravity
flow (see Note 5).
(a) Take a sample of the eluate (E) from each strain for
denaturing SDS-PAGE evaluation.
15. Record the absorption spectrum from 200 to 350 nm and
determine the concentration of AP205 VLPs taking into
account the protein and nucleic acid concentration according
to the algorithm of Porterfield and Zlotnick [12].
16. Concentrate and buffer exchange the purified proteins into
freezing buffer using 100 kDa MWCO centrifugal filters for
an approximate final concentration of 1–2 mg/mL (see Note
6).
17. After protein recovery from concentrators, centrifuge samples
at 20,000 Â g for 15 min to remove aggregates. Discard pellet.
18. Purified protein is stable at 4
C for at least 6 months or at
À80
C for years.
3.4 Analysis
of Purified VLPs
1. AP205 VLPs can be evaluated for particle assembly by native
electrophoresis on a 0.6% TAE-agarose gel (see Note 7). Fully
assembled particles are observed as distinct bands which stain
for both nucleic acids and protein (Fig. 2a).
2. Particle purity and glycosylation status can be qualitatively
evaluated by SDS-PAGE with Coomassie staining (Fig. 2b)
(see Notes 8 and 9).
3. Intact protein mass spectrometry can be used to semiquantitatively evaluate glycosylation of the coat proteins (Fig. 3).
4. Transmission electron microscopy can be used to visualize the
particle structure (Fig. 4) (see Note 10).
4 Notes
1. In the A. pleuropneumoniae genome, ORFs encoding the NGT
and α6GlcT are arranged sequentially as part of a polycistronic
operon. Previous studies have demonstrated that it is possible
to amplify these two ORFs and express them in a bicistronic
arrangement under the control of a single promoter [4].
Biosynthesis of Glycoconjugate VLPs
211