6. As a step-wise alternative, one could first buffer exchange by
dialysis overnight and concentrate the proteins using 100 kDa
MWCO centrifugal filters the next day.
7. AP205 VLPs can be electrophoresed on agarose gels with the
same buffers, reagents, and gel documentation system used for
nucleic acid analysis. In our experience, separation of 5–10 μg
of purified VLP, on a 0.6% agarose gel, with TAE buffer,
Fig. 3 AP205cp-GS is glycosylated by the NGT and α6GlcT. Purified VLPs were reduced with DTT, desalted
using a C4 ZipTip, and analyzed by electrospray-TOF mass spectrometry. Spectra were acquired in positiveion mode. The m/z data were deconvoluted into mass spectra with a resolution of 0.5 Da/channel
Fig. 4 Glycosylated AP205cp-GS assembles into 30 nm VLPs, as shown after expression without glycosyltransferases (left panel), coexpression with the NGT (middle panel) and with the NGT and α6GlcT (right panel).
Purified VLP samples were adsorbed onto carbon-coated copper grids and stained with 2% (w/v) uranyl
acetate (pH 4). Grids were examined with a transmission electron microscope fitted with a 100 kV tungsten
emitter and 1376 Â 1032 pixel CCD detector
Biosynthesis of Glycoconjugate VLPs
213
dialysis overnight and concentrate the proteins using 100 kDa
MWCO centrifugal filters the next day.
7. AP205 VLPs can be electrophoresed on agarose gels with the
same buffers, reagents, and gel documentation system used for
nucleic acid analysis. In our experience, separation of 5–10 μg
of purified VLP, on a 0.6% agarose gel, with TAE buffer,
Fig. 3 AP205cp-GS is glycosylated by the NGT and α6GlcT. Purified VLPs were reduced with DTT, desalted
using a C4 ZipTip, and analyzed by electrospray-TOF mass spectrometry. Spectra were acquired in positiveion mode. The m/z data were deconvoluted into mass spectra with a resolution of 0.5 Da/channel
Fig. 4 Glycosylated AP205cp-GS assembles into 30 nm VLPs, as shown after expression without glycosyltransferases (left panel), coexpression with the NGT (middle panel) and with the NGT and α6GlcT (right panel).
Purified VLP samples were adsorbed onto carbon-coated copper grids and stained with 2% (w/v) uranyl
acetate (pH 4). Grids were examined with a transmission electron microscope fitted with a 100 kV tungsten
emitter and 1376 Â 1032 pixel CCD detector
Biosynthesis of Glycoconjugate VLPs
213