2.2 Protein
Expression Using
Brevibacillus Cells
1. Brevibacillus choshinensis HPD31-SP3 (Takara Bio/Clontech).
2. TM medium: 10 g/L glucose, 10 g/L polypeptone, 5 g/L
meat extract, 2 g/L yeast extract, 10 mg/L FeSO 4 Á7H 2 O,
10 mg/L MnSO 4 Á4H 2 O, 1 mg/L ZnSO 4 Á7H 2 O, pH 7.0.
3. MT medium: 10 g/L glucose, 10 g/L polypeptone, 5 g/L
meat extract, 2 g/L yeast extract, 10 mg/L FeSO 4 Á7H 2 O,
10 mg/L MnSO 4 Á4H 2 O, 1 mg/L ZnSO 4 Á7H 2 O, 4.1 g/L
MgCl 2 , pH 7.0.
4. MT/neomycin agar plate: 10 g/L glucose, 10 g/L polypeptone, 5 g/L meat extract, 2 g/L yeast extract, 10 mg/L
FeSO 4 Á7H 2 O, 10 mg/L MnSO 4 Á4H 2 O, 1 mg/L
ZnSO 4 Á7H 2 O, 4.1 g/L MgCl 2 , 10 mg/L neomycin, 15 g/L
agar, pH 7.0.
5. 2SY/neomycin medium: 20 g/L glucose, 40 g/L Bacto Soytone, 5 g/L yeast extract, 0.15 g/L CaCl 2 Á2H 2 O, 50 mg/L
neomycin.
6. PGH buffer: 1 mM HEPES-KOH (pH 7.0), 15% (w/v) glycerol, 15% (w/v) PEG6000.
7. pBIM2 vector (Fig. 2).
8. Synthetic or natural antibody-encoding cDNA of interest (see
Note 1).
9. BIM2-F primer: 5
0 -ACTAGTGAAAATTTATATTTTCAAGG
TCACCATCACCATCACCATTCCTCTGGTGGCGGT.
10. BIM2-R primer: 5
0 -AAGCTTCGATTGGAAGTACAGGTTT
TCGGAAGATCTAGAGGAACCACCCCCACCCCCGAG.
11. pNY326-F primer: 5
0 -GAATTCGGTACCCCGGGTTCGA.
12. pNY326-R
primer:
5
0 -
GGATCCTGCAGCGAAAGCCATGGGAGC.
13. BacillusExp-F primer: 5
0 -CACGCGCTTGCAGGATTCGG.
14. BacillusExp-R primer: 5
0 - CAATGTAATTGTTCCCTACCT
GC.
15. GenePulser electroporator (Bio-Rad).
16. 0.2-cm electroporation cuvette.
17. 125-mL baffled bottom Erlenmeyer flask.
18. 500-mL baffled bottom Erlenmeyer flask.
19. 2.5-L Tunair baffled shaker flask.
20. Orbital shaker incubator.
21. Centrifuge with adaptors for 1-L, 250-mL, 50-mL, and 15-mL
tubes.
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Norimichi Nomura et al.