2. We found C-terminal tags with less than a nine-amino acid
spacer to be less efficiently glycosylated, because the VLP surface sterically hinders NGT access to the glycosite.
3. The affinity tag, Strep-tag II, is not necessary for glycosylation
and may be removed if purification via precipitation or sizeexclusion chromatography is desired.
4. Settings apply to Qsonica Sonicators. Alternatively, cells can
also be lysed using a French pressure cell press. We observed
efficient lysis with three passages through the cell press at
800–1200 PSIG without lysozyme treatment.
5. Allow proteins to unbind from the Strep-Tactin beads with a
10 min incubation in elution buffer before eluting. Yields may
be improved by eluting with 1 CV and repeating it four times.
Pool the eluate fractions.
Fig. 2 Native and denaturing electrophoresis of purified VLPs. The AP205cp-GS
construct was expressed alone, or in combination with the NGT and/or α6GlcT
(as indicated above the gel images), then purified by affinity chromatography. (a)
10 μg of each purified protein was loaded on a 0.6% TAE agarose gel, containing
0.5 μg/mL ethidium bromide. Samples were electrophoresed for 40 min at 120 V
in a TAE bath. The gel was imaged under UV illumination. Ethidium bromide
binds to nucleic acids encapsulated in the VLPs. (b) Samples of each purified
protein were denatured at 95
C in reducing Laemmli buffer, then 3 μg of each
was separated on 13% tricine SDS-PAGE and proteins were visualized by
Coomassie staining
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Kathryn K. Oi et al.
spacer to be less efficiently glycosylated, because the VLP surface sterically hinders NGT access to the glycosite.
3. The affinity tag, Strep-tag II, is not necessary for glycosylation
and may be removed if purification via precipitation or sizeexclusion chromatography is desired.
4. Settings apply to Qsonica Sonicators. Alternatively, cells can
also be lysed using a French pressure cell press. We observed
efficient lysis with three passages through the cell press at
800–1200 PSIG without lysozyme treatment.
5. Allow proteins to unbind from the Strep-Tactin beads with a
10 min incubation in elution buffer before eluting. Yields may
be improved by eluting with 1 CV and repeating it four times.
Pool the eluate fractions.
Fig. 2 Native and denaturing electrophoresis of purified VLPs. The AP205cp-GS
construct was expressed alone, or in combination with the NGT and/or α6GlcT
(as indicated above the gel images), then purified by affinity chromatography. (a)
10 μg of each purified protein was loaded on a 0.6% TAE agarose gel, containing
0.5 μg/mL ethidium bromide. Samples were electrophoresed for 40 min at 120 V
in a TAE bath. The gel was imaged under UV illumination. Ethidium bromide
binds to nucleic acids encapsulated in the VLPs. (b) Samples of each purified
protein were denatured at 95
C in reducing Laemmli buffer, then 3 μg of each
was separated on 13% tricine SDS-PAGE and proteins were visualized by
Coomassie staining
212
Kathryn K. Oi et al.