glycerol stock tube at À80
C. The stock is now stable for
years, as long as it is kept at À80
C.
27. Extract and purify the plasmid DNA from the remaining 5 mL
of each culture using the QIAquick spin miniprep kit, according to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer.
28. Using the vector primers, BacillusExp-F and BacillusExp-R,
perform sequence analysis to confirm that the nucleotide
sequence of the plasmids is correct.
3.1.3 Secretory
Expression Using
Brevibacillus Cells
1. Day 1: (Pre-culture) Open the tube of the glycerol stock of
Brevibacillus choshinensis harboring a positive pBIM2 expression plasmid and scrape some of the frozen bacteria off using a
sterile loop. Inoculate the bacteria into 50 mL of 2SY/neomycin medium in a 125-mL baffled-bottom Erlenmeyer flask and
grow for 40–55 h at 30
C and 200 rpm.
2. Day 3: (Large-scale culture) Inoculate 1 L of 2SY/neomycin
medium into a 2.5-L Tunair baffled shaker flask with 10 mL of
saturated pre-culture.
3. Grow the cells for 48–60 h at 30
C and 200 rpm.
4. Day 5: Centrifuge the culture at 6000 Â g for 15 min at 4
C.
Recover the supernatant and discard the cell pellet.
3.1.4 Purification
of Tag-Free Fv Fragments
1. Day 1: (Ammonium sulfate precipitation) To the 1 L of Brevibacillus culture supernatant, add 390 g of ammonium sulfate
powder slowly but steadily with thorough mixing to adjust to a
final concentration of 60% saturation. Do not allow clumps
to form.
2. Allow a precipitate to form for 1 h at 4
C with stirring.
3. Recover the precipitate by centrifugation at 10,000 Â g for
20 min at 4
C.
4. Dissolve the precipitate by adding 10 mL of TBS.
5. Dialyze overnight against 1 L of TBS at 4
C.
6. Centrifuge the dialyzed sample at 15,000 Â g for 10 min at
4
C. Recover the supernatant and discard the precipitate.
7. Day 2: (Immobilized metal affinity chromatography (IMAC))
Use 5 mL of Ni-NTA resin (10 mL of 50% slurry) per 1 L of
Brevibacillus culture, pour the slurry into a glass EconoColumn, and equilibrate with five column volumes (CVs) of
buffer A.
8. Add the supernatant to the column and collect the flowthrough fraction.
9. Wash the column with 10 CVs (50 mL) of buffer A.
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Norimichi Nomura et al.
C. The stock is now stable for
years, as long as it is kept at À80
C.
27. Extract and purify the plasmid DNA from the remaining 5 mL
of each culture using the QIAquick spin miniprep kit, according to the manufacturer’s instructions. Measure the DNA concentration using a NanoDrop spectrophotometer.
28. Using the vector primers, BacillusExp-F and BacillusExp-R,
perform sequence analysis to confirm that the nucleotide
sequence of the plasmids is correct.
3.1.3 Secretory
Expression Using
Brevibacillus Cells
1. Day 1: (Pre-culture) Open the tube of the glycerol stock of
Brevibacillus choshinensis harboring a positive pBIM2 expression plasmid and scrape some of the frozen bacteria off using a
sterile loop. Inoculate the bacteria into 50 mL of 2SY/neomycin medium in a 125-mL baffled-bottom Erlenmeyer flask and
grow for 40–55 h at 30
C and 200 rpm.
2. Day 3: (Large-scale culture) Inoculate 1 L of 2SY/neomycin
medium into a 2.5-L Tunair baffled shaker flask with 10 mL of
saturated pre-culture.
3. Grow the cells for 48–60 h at 30
C and 200 rpm.
4. Day 5: Centrifuge the culture at 6000 Â g for 15 min at 4
C.
Recover the supernatant and discard the cell pellet.
3.1.4 Purification
of Tag-Free Fv Fragments
1. Day 1: (Ammonium sulfate precipitation) To the 1 L of Brevibacillus culture supernatant, add 390 g of ammonium sulfate
powder slowly but steadily with thorough mixing to adjust to a
final concentration of 60% saturation. Do not allow clumps
to form.
2. Allow a precipitate to form for 1 h at 4
C with stirring.
3. Recover the precipitate by centrifugation at 10,000 Â g for
20 min at 4
C.
4. Dissolve the precipitate by adding 10 mL of TBS.
5. Dialyze overnight against 1 L of TBS at 4
C.
6. Centrifuge the dialyzed sample at 15,000 Â g for 10 min at
4
C. Recover the supernatant and discard the precipitate.
7. Day 2: (Immobilized metal affinity chromatography (IMAC))
Use 5 mL of Ni-NTA resin (10 mL of 50% slurry) per 1 L of
Brevibacillus culture, pour the slurry into a glass EconoColumn, and equilibrate with five column volumes (CVs) of
buffer A.
8. Add the supernatant to the column and collect the flowthrough fraction.
9. Wash the column with 10 CVs (50 mL) of buffer A.
88
Norimichi Nomura et al.