3 Methods
3.1 Original iRAT
System
for the Production
of Recombinant Fv
Fragments
Fv fragments (with a molecular weight of ~25 kDa) are heterodimeric molecules consisting of two domains, V L and V H , that assemble through hydrophobic interactions. Each of the V L and V H
domains contains an intrachain disulfide bond for stabilization.
These structural properties require a sophisticated apparatus for
secretion, folding, and assembly, as well as an oxidizing environment for disulfide bond generation. The expression of Fv fragments
via the B. choshinensis Sec-dependent secretion pathway ensures the
formation of the intrachain disulfide bonds to stabilize the structure. A reason to include MBP in the iRAT segment is that the
distance between the N- and C-termini (43 A ˚ ) is similar to the
distance between the C-terminus of V L and the N-terminus of V H
(approximately 35–40 A ˚ ) for most Fv fragments.
3.1.1 Preparation
of Brevibacillus
Electrocompetent Cells
1. Day 1: Inoculate a single colony of B. choshinensis into 10 mL of
TM medium and grow overnight at 37
C and 200 rpm.
2. Day 2: Inoculate 100 mL of TM medium in a baffled bottom
500-mL Erlenmeyer flask with 1 mL of overnight culture and
incubate at 37
C and 200 rpm until an OD 600nm of 3.0–4.0
(see Note 3).
3. Centrifuge at 5000 Â g for 10 min at 4
C.
4. Resuspend the cell pellet in 20 mL of PGH buffer.
5. Centrifuge at 8000 Â g for 20 min at 4
C.
6. Remove the supernatant thoroughly and resuspend the cell
pellet carefully in 2 mL of PGH buffer.
7. Dispense into 100 μL aliquots and flash-freeze with liquid
nitrogen.
8. Store at À80
C.
3.1.2 Construction
of the Brevibacillus
Expression Plasmid
The pBIM2 vector (Fig. 2a) for the expression of Fv fragments is
designed based on the backbone of the Brevibacillus plasmid
pNY326. The V L -insertion site, iRAT cassette, and V H -insertion
site are tandemly arranged in this order and are introduced downstream of the constitutively active P5 promoter and Sec secretion
signal sequence. The iRAT-MBP cassette encodes the N-terminal
TEV cleavage site, His 6 tag, a 9-residue Gly/Ser-rich linker, the
MBP coding sequence (residues 27–392 from UniProt ID:
P0AEX9), a 25-residue Asn/Gly/Ser-rich linker, and a
C-terminal TEV cleavage site (Fig. 2c). Residues 1–107 (Kabat
numbering scheme) of the V L domain and 1–113 of the V H domain
derived from monoclonal antibodies are used for Fv fragment
expression.
84
Norimichi Nomura et al.
3.1 Original iRAT
System
for the Production
of Recombinant Fv
Fragments
Fv fragments (with a molecular weight of ~25 kDa) are heterodimeric molecules consisting of two domains, V L and V H , that assemble through hydrophobic interactions. Each of the V L and V H
domains contains an intrachain disulfide bond for stabilization.
These structural properties require a sophisticated apparatus for
secretion, folding, and assembly, as well as an oxidizing environment for disulfide bond generation. The expression of Fv fragments
via the B. choshinensis Sec-dependent secretion pathway ensures the
formation of the intrachain disulfide bonds to stabilize the structure. A reason to include MBP in the iRAT segment is that the
distance between the N- and C-termini (43 A ˚ ) is similar to the
distance between the C-terminus of V L and the N-terminus of V H
(approximately 35–40 A ˚ ) for most Fv fragments.
3.1.1 Preparation
of Brevibacillus
Electrocompetent Cells
1. Day 1: Inoculate a single colony of B. choshinensis into 10 mL of
TM medium and grow overnight at 37
C and 200 rpm.
2. Day 2: Inoculate 100 mL of TM medium in a baffled bottom
500-mL Erlenmeyer flask with 1 mL of overnight culture and
incubate at 37
C and 200 rpm until an OD 600nm of 3.0–4.0
(see Note 3).
3. Centrifuge at 5000 Â g for 10 min at 4
C.
4. Resuspend the cell pellet in 20 mL of PGH buffer.
5. Centrifuge at 8000 Â g for 20 min at 4
C.
6. Remove the supernatant thoroughly and resuspend the cell
pellet carefully in 2 mL of PGH buffer.
7. Dispense into 100 μL aliquots and flash-freeze with liquid
nitrogen.
8. Store at À80
C.
3.1.2 Construction
of the Brevibacillus
Expression Plasmid
The pBIM2 vector (Fig. 2a) for the expression of Fv fragments is
designed based on the backbone of the Brevibacillus plasmid
pNY326. The V L -insertion site, iRAT cassette, and V H -insertion
site are tandemly arranged in this order and are introduced downstream of the constitutively active P5 promoter and Sec secretion
signal sequence. The iRAT-MBP cassette encodes the N-terminal
TEV cleavage site, His 6 tag, a 9-residue Gly/Ser-rich linker, the
MBP coding sequence (residues 27–392 from UniProt ID:
P0AEX9), a 25-residue Asn/Gly/Ser-rich linker, and a
C-terminal TEV cleavage site (Fig. 2c). Residues 1–107 (Kabat
numbering scheme) of the V L domain and 1–113 of the V H domain
derived from monoclonal antibodies are used for Fv fragment
expression.
84
Norimichi Nomura et al.