10. Following induction, continue incubation at 28
C, with
shaking at 180 rpm overnight (18–22 h) prior to harvest.
Day 4: Harvest Cells
11. Place cultures on ice and measure OD 600 .
12. Transfer the cooled cultures into centrifuge tubes and spin
down the cells at 8000 Â g, at 4
C, for 20 min. Discard the
supernatant.
13. Resuspend the cells in 40 mL of cold PBS and transfer the
cells into new tubes.
14. Spin the cells at 4000 Â g at 4
C for 15 min. Discard the
supernatant.
15. Determine the approximate wet weights of the cell pellets by
weighing against an empty tube.
16. Proceed to cell lysis or freeze the pellets in liquid nitrogen and
store them at À20
C.
3.3 Purification
of Glycoconjugate
AP205 VLPs
Day 5: Cell lysis by sonication and lysozyme treatment
1. Resuspend the cell pellets in lysis solution (approximately 5 mL
per 1 g of pellet).
2. Incubate at 37
C with mixing for 2 h.
3. Cool the lysates on ice.
4. Disrupt the cells by sonication using a 4 mm tip, total process
time of 90 s (10 s pulse, 5 s pause), and amplitude 40 (see Note
4).
(a) Take a sample of each lysate as whole cell extract (WCE)
for denaturing SDS-PAGE evaluation.
5. Pellet unlysed cells and cellular debris by centrifugation at
25,000 Â g for 30 min, 4
C.
6. Transfer supernatant to new tube (pellet can be discarded).
(a) Take a sample of each supernatant as soluble fraction
(S) for denaturing SDS-PAGE evaluation.
7. Proceed to protein purification or freeze lysates in liquid nitrogen and store at À20
C.
Day 6: Protein purification
8. Preequilibrate 5 mL of Strep-Tactin Superflow beads per sample; wash the beads twice with 10 column volumes (CV) of
binding buffer.
9. Spin down beads at 300 Â g for 2 min and discard supernatant.
10. Add the soluble protein lysates to the Strep-Tactin Superflow
beads, and incubate at 25
C for 60 min on a rotator.
210
Kathryn K. Oi et al.
C, with
shaking at 180 rpm overnight (18–22 h) prior to harvest.
Day 4: Harvest Cells
11. Place cultures on ice and measure OD 600 .
12. Transfer the cooled cultures into centrifuge tubes and spin
down the cells at 8000 Â g, at 4
C, for 20 min. Discard the
supernatant.
13. Resuspend the cells in 40 mL of cold PBS and transfer the
cells into new tubes.
14. Spin the cells at 4000 Â g at 4
C for 15 min. Discard the
supernatant.
15. Determine the approximate wet weights of the cell pellets by
weighing against an empty tube.
16. Proceed to cell lysis or freeze the pellets in liquid nitrogen and
store them at À20
C.
3.3 Purification
of Glycoconjugate
AP205 VLPs
Day 5: Cell lysis by sonication and lysozyme treatment
1. Resuspend the cell pellets in lysis solution (approximately 5 mL
per 1 g of pellet).
2. Incubate at 37
C with mixing for 2 h.
3. Cool the lysates on ice.
4. Disrupt the cells by sonication using a 4 mm tip, total process
time of 90 s (10 s pulse, 5 s pause), and amplitude 40 (see Note
4).
(a) Take a sample of each lysate as whole cell extract (WCE)
for denaturing SDS-PAGE evaluation.
5. Pellet unlysed cells and cellular debris by centrifugation at
25,000 Â g for 30 min, 4
C.
6. Transfer supernatant to new tube (pellet can be discarded).
(a) Take a sample of each supernatant as soluble fraction
(S) for denaturing SDS-PAGE evaluation.
7. Proceed to protein purification or freeze lysates in liquid nitrogen and store at À20
C.
Day 6: Protein purification
8. Preequilibrate 5 mL of Strep-Tactin Superflow beads per sample; wash the beads twice with 10 column volumes (CV) of
binding buffer.
9. Spin down beads at 300 Â g for 2 min and discard supernatant.
10. Add the soluble protein lysates to the Strep-Tactin Superflow
beads, and incubate at 25
C for 60 min on a rotator.
210
Kathryn K. Oi et al.