2. Design of a glycosylation competent VLP: In this protocol, we
target the coat protein of the AP205 bacteriophage for glycosylation. This is achieved by genetic fusion of a short polypeptide tag including a glycosylation site (GS) to the C-terminus of
the AP205cp. The tag provides a spacer of 9 amino acids
between the native C-terminus of the coat protein and the
glycosylation site (see Note 2). The tag includes an extended
glycosylation site (ANATA) based on a consensus sequence
determined for the NGT [5]. Finally the tag includes a Streptag II sequence (WSHPQFEK) for affinity purification [11] (see
Note 3).
3.2 Expression
of Glycoconjugate
VLPs
Day 1: Transformation
1. For each strain, dilute approximately 50 ng of each plasmid into
2 μL ddH 2 O. Mix with 50 μL of electrocompetent E. coli
BL21-Gold(DE3) and transfer to a cold 1 mm electroporation
cuvette on ice. Proceed immediately to electroporation.
(a) Strain A: pRSF_AP205cp-GS.
(b) Strain B: pRSF_AP205cp-GS + pACYC_NGT.
(c) Strain C: pRSF_AP205cp-GS + pACYC_NGT + pC
DF_α6GlcT.
2. Electroporate cells using the following parameters: 1800 V,
200 Ω, 25 μF.
3. Directly transfer electroporated cells to 1 mL of LB and allow
to recover for 1 h at 37
C.
4. Pellet cells at 8000 Â g for 3 min, discard 950 μL of supernatant, resuspend cells in the remaining media.
5. Plate cells on LB agar with the necessary antibiotic(s) and
incubate overnight at 37
C.
Day 2: Precultures
6. Inoculate 10 mL of LB medium with antibiotic(s) with multiple colonies and grow overnight at 37
C.
Day 3: Protein Expression Cultures
7. Transfer 200 mL of TB medium, containing necessary antibiotic(s), into a 1 L Erlenmeyer flask and inoculate with preculture to an OD 600 of 0.05.
8. Place the expression culture in 37
C incubator with shaking at
180 rpm and measure OD 600 at half-hourly intervals.
9. When OD 600 reaches 0.6–1.0, cool the cultures to below
28
C, then induce protein expression by addition of
1 mM IPTG.
Biosynthesis of Glycoconjugate VLPs
209
target the coat protein of the AP205 bacteriophage for glycosylation. This is achieved by genetic fusion of a short polypeptide tag including a glycosylation site (GS) to the C-terminus of
the AP205cp. The tag provides a spacer of 9 amino acids
between the native C-terminus of the coat protein and the
glycosylation site (see Note 2). The tag includes an extended
glycosylation site (ANATA) based on a consensus sequence
determined for the NGT [5]. Finally the tag includes a Streptag II sequence (WSHPQFEK) for affinity purification [11] (see
Note 3).
3.2 Expression
of Glycoconjugate
VLPs
Day 1: Transformation
1. For each strain, dilute approximately 50 ng of each plasmid into
2 μL ddH 2 O. Mix with 50 μL of electrocompetent E. coli
BL21-Gold(DE3) and transfer to a cold 1 mm electroporation
cuvette on ice. Proceed immediately to electroporation.
(a) Strain A: pRSF_AP205cp-GS.
(b) Strain B: pRSF_AP205cp-GS + pACYC_NGT.
(c) Strain C: pRSF_AP205cp-GS + pACYC_NGT + pC
DF_α6GlcT.
2. Electroporate cells using the following parameters: 1800 V,
200 Ω, 25 μF.
3. Directly transfer electroporated cells to 1 mL of LB and allow
to recover for 1 h at 37
C.
4. Pellet cells at 8000 Â g for 3 min, discard 950 μL of supernatant, resuspend cells in the remaining media.
5. Plate cells on LB agar with the necessary antibiotic(s) and
incubate overnight at 37
C.
Day 2: Precultures
6. Inoculate 10 mL of LB medium with antibiotic(s) with multiple colonies and grow overnight at 37
C.
Day 3: Protein Expression Cultures
7. Transfer 200 mL of TB medium, containing necessary antibiotic(s), into a 1 L Erlenmeyer flask and inoculate with preculture to an OD 600 of 0.05.
8. Place the expression culture in 37
C incubator with shaking at
180 rpm and measure OD 600 at half-hourly intervals.
9. When OD 600 reaches 0.6–1.0, cool the cultures to below
28
C, then induce protein expression by addition of
1 mM IPTG.
Biosynthesis of Glycoconjugate VLPs
209