supplemented with 0.25 μg/mL ethidium bromide, is ideal for
separation and visualization of VLPs. Ethidium bromide labels
nucleic acids encapsulated within the VLPs. The protein component of VLPs can subsequently be stained by placing the
agarose gel in Colloidal Coomassie Staining solution (see
Note 8).
8. Increased sensitivity and reduced background staining of agarose and polyacrylamide gels, is achieved with the Colloidal
Coomassie Staining and Destaining Solutions developed by
Kang and colleagues [13].
9. To separate different glycoforms of AP205cp-GS, we recommend 13% tricine SDS-PAGE gels as described by
Sch€ agger [14].
10. For best results, 400-mesh carbon-coated copper grids are
negatively charged in an oxygen plasma, then placed facedown on a 5 μL droplet of 20 nM VLP for 60 s. Adsorbed
particles are stained with 2% (w/v) uranyl acetate (pH 4) for
30 s, then imaged.
Acknowledgments
We are grateful to Professor Markus Aebi for his guidance in study
design and comments on the manuscript, Dr. Miriam Lucas and
ScopeM for assistance with transmission electron microscopy, and
Dr. Serge Chesnov and the FGCZ for mass spectrometric analysis.
This research was funded through a Bridge Discovery grant and by an
ETH Zurich Career Seed Grant (SEED-33 16-1) awarded to T.G.K.
References
1. Ramjeet M, Deslandes V, Goure J, Jacques M
(2008) Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies. Anim Health Res Rev 9
(1):25–45
2. Choi KJ, Grass S, Paek S, St Geme JW 3rd, Yeo
HJ (2010) The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates
N-linked glycosylation of the Haemophilus
influenzae HMW1 adhesin. PLoS One 5(12):
e15888
3. Schwarz F, Fan YY, Schubert M, Aebi M
(2011) Cytoplasmic N-glycosyltransferase of
Actinobacillus pleuropneumoniae is an inverting enzyme and recognizes the NX(S/T) consensus sequence. J Biol Chem 286
(40):35267–35274
4. Cuccui J, Terra VS, Bosse JT, Naegeli A,
Abouelhadid S, Li Y, Lin CW, Vohra P, Tucker
AW, Rycroft AN, Maskell DJ, Aebi M, Langford PR, Wren BW, Consortium BRT (2017)
The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for
adhesion and has potential use in glycoengineering. Open Biol 7(1)
5. Naegeli A, Neupert C, Fan YY, Lin CW,
Poljak K, Papini AM, Schwarz F, Aebi M
(2014) Molecular analysis of an alternative
N-glycosylation machinery by functional transfer from Actinobacillus pleuropneumoniae to
Escherichia coli. J Biol Chem 289
(4):2170–2179
6. Keys TG, Wetter M, Hang I, Rutschmann C,
Russo S, Mally M, Steffen M, Zuppiger M,
Muller F, Schneider J, Faridmoayer A, Lin
CW, Aebi M (2017) A biosynthetic route for
polysialylating proteins in Escherichia coli.
Metab Eng 44:293–301
214
Kathryn K. Oi et al.
separation and visualization of VLPs. Ethidium bromide labels
nucleic acids encapsulated within the VLPs. The protein component of VLPs can subsequently be stained by placing the
agarose gel in Colloidal Coomassie Staining solution (see
Note 8).
8. Increased sensitivity and reduced background staining of agarose and polyacrylamide gels, is achieved with the Colloidal
Coomassie Staining and Destaining Solutions developed by
Kang and colleagues [13].
9. To separate different glycoforms of AP205cp-GS, we recommend 13% tricine SDS-PAGE gels as described by
Sch€ agger [14].
10. For best results, 400-mesh carbon-coated copper grids are
negatively charged in an oxygen plasma, then placed facedown on a 5 μL droplet of 20 nM VLP for 60 s. Adsorbed
particles are stained with 2% (w/v) uranyl acetate (pH 4) for
30 s, then imaged.
Acknowledgments
We are grateful to Professor Markus Aebi for his guidance in study
design and comments on the manuscript, Dr. Miriam Lucas and
ScopeM for assistance with transmission electron microscopy, and
Dr. Serge Chesnov and the FGCZ for mass spectrometric analysis.
This research was funded through a Bridge Discovery grant and by an
ETH Zurich Career Seed Grant (SEED-33 16-1) awarded to T.G.K.
References
1. Ramjeet M, Deslandes V, Goure J, Jacques M
(2008) Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies. Anim Health Res Rev 9
(1):25–45
2. Choi KJ, Grass S, Paek S, St Geme JW 3rd, Yeo
HJ (2010) The Actinobacillus pleuropneumoniae HMW1C-like glycosyltransferase mediates
N-linked glycosylation of the Haemophilus
influenzae HMW1 adhesin. PLoS One 5(12):
e15888
3. Schwarz F, Fan YY, Schubert M, Aebi M
(2011) Cytoplasmic N-glycosyltransferase of
Actinobacillus pleuropneumoniae is an inverting enzyme and recognizes the NX(S/T) consensus sequence. J Biol Chem 286
(40):35267–35274
4. Cuccui J, Terra VS, Bosse JT, Naegeli A,
Abouelhadid S, Li Y, Lin CW, Vohra P, Tucker
AW, Rycroft AN, Maskell DJ, Aebi M, Langford PR, Wren BW, Consortium BRT (2017)
The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for
adhesion and has potential use in glycoengineering. Open Biol 7(1)
5. Naegeli A, Neupert C, Fan YY, Lin CW,
Poljak K, Papini AM, Schwarz F, Aebi M
(2014) Molecular analysis of an alternative
N-glycosylation machinery by functional transfer from Actinobacillus pleuropneumoniae to
Escherichia coli. J Biol Chem 289
(4):2170–2179
6. Keys TG, Wetter M, Hang I, Rutschmann C,
Russo S, Mally M, Steffen M, Zuppiger M,
Muller F, Schneider J, Faridmoayer A, Lin
CW, Aebi M (2017) A biosynthetic route for
polysialylating proteins in Escherichia coli.
Metab Eng 44:293–301
214
Kathryn K. Oi et al.