2.3 Protein
Expression Using Sf9
Cells
and Baculoviruses
1. E. coli TOP10 chemically competent cells (Thermo Fisher
Scientific).
2. E. coli DH10Bac chemically competent cells (Thermo Fisher
Scientific).
3. SOC medium: 20 g/L tryptone, 5 g/L yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM
glucose.
4. LB/ampicillin plate: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 100 mg/L ampicillin, 15 g/L agar.
5. LB/ampicillin medium: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 100 mg/L ampicillin.
6. LB/KTGIB agar plate: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 50 mg/L kanamycin, 10 mg/L tetracycline,
7 mg/L gentamicin, 40 mg/L IPTG, 50 mg/L X-Gal, 15 g/
L agar.
7. LB/KTG medium: 10 g/L tryptone, 5 g/L yeast extract, 5 g/
L NaCl, 50 mg/L kanamycin, 10 mg/L tetracycline, 7 mg/L
gentamicin.
8. pFBGP67-iRATmC (Fig. 4).
9. IRAT70-F primer: 5
0 -ACTAGTGAAAATTTATATTTTCAAG
GTCACCATCACCATCACCATGGTGGCTCCTCTGGT.
10. IRAT70-R primer: 5
0 - AAGCTTCGATTGGAAGTACA
GGTTTTCACCACCACTACTACTGGAACCCGAAGAGC
CGGA.
11. FB-F1 primer: 5
0 - TAAAAGCTTGTCGAGAAGTACTAGAG
GATCATAATCAGCCATACCACA.
12. gp67-R primer: 5
0 - CGCCGCAAAGGCAGAATGCGCCGC
CGCCGCCAAAAGCACATATAAAAC.
13. SFseq-F primer: 5
0 -ACTGTTTTCGTAACAGTTTTGTAATA.
14. SFseq-R primer: 5
0 - GTGGTATGGCTGATTATGATCCTCT
AGTACTTCT.
15. Sf9 cells adapted for suspension growth.
16. PSFM-J1 medium (Wako).
17. Fetal bovine serum (FBS).
ä
Fig. 2 (continued) polyprotein construct. (b) Universal PCR primers for the
construction of expression plasmid. (c) Sequence corresponding to the region
shown in (a). P5–35 and P5–10 represent the À35 and À10 box of the promoter
P5, respectively. SD1 and SD2 are Shine-Dalgarno sequences. The Sec secretion signal, TEV recognition sequence, His 6 tag, and MBP are shown in gray,
orange, yellow, and green, respectively. Gray arrowheads indicate the signal
cleavage site and TEV cleavage sites
82
Norimichi Nomura et al.
Expression Using Sf9
Cells
and Baculoviruses
1. E. coli TOP10 chemically competent cells (Thermo Fisher
Scientific).
2. E. coli DH10Bac chemically competent cells (Thermo Fisher
Scientific).
3. SOC medium: 20 g/L tryptone, 5 g/L yeast extract, 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 , 20 mM
glucose.
4. LB/ampicillin plate: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 100 mg/L ampicillin, 15 g/L agar.
5. LB/ampicillin medium: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 100 mg/L ampicillin.
6. LB/KTGIB agar plate: 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, 50 mg/L kanamycin, 10 mg/L tetracycline,
7 mg/L gentamicin, 40 mg/L IPTG, 50 mg/L X-Gal, 15 g/
L agar.
7. LB/KTG medium: 10 g/L tryptone, 5 g/L yeast extract, 5 g/
L NaCl, 50 mg/L kanamycin, 10 mg/L tetracycline, 7 mg/L
gentamicin.
8. pFBGP67-iRATmC (Fig. 4).
9. IRAT70-F primer: 5
0 -ACTAGTGAAAATTTATATTTTCAAG
GTCACCATCACCATCACCATGGTGGCTCCTCTGGT.
10. IRAT70-R primer: 5
0 - AAGCTTCGATTGGAAGTACA
GGTTTTCACCACCACTACTACTGGAACCCGAAGAGC
CGGA.
11. FB-F1 primer: 5
0 - TAAAAGCTTGTCGAGAAGTACTAGAG
GATCATAATCAGCCATACCACA.
12. gp67-R primer: 5
0 - CGCCGCAAAGGCAGAATGCGCCGC
CGCCGCCAAAAGCACATATAAAAC.
13. SFseq-F primer: 5
0 -ACTGTTTTCGTAACAGTTTTGTAATA.
14. SFseq-R primer: 5
0 - GTGGTATGGCTGATTATGATCCTCT
AGTACTTCT.
15. Sf9 cells adapted for suspension growth.
16. PSFM-J1 medium (Wako).
17. Fetal bovine serum (FBS).
ä
Fig. 2 (continued) polyprotein construct. (b) Universal PCR primers for the
construction of expression plasmid. (c) Sequence corresponding to the region
shown in (a). P5–35 and P5–10 represent the À35 and À10 box of the promoter
P5, respectively. SD1 and SD2 are Shine-Dalgarno sequences. The Sec secretion signal, TEV recognition sequence, His 6 tag, and MBP are shown in gray,
orange, yellow, and green, respectively. Gray arrowheads indicate the signal
cleavage site and TEV cleavage sites
82
Norimichi Nomura et al.