fluorescent Fab with complete target-binding activity before
removal of the iRAT segment as described in the Subheading
3.2.3 (see Fig. 5). Once the iRAT segment is excised by TEV
protease, a large amount of tag-free Fab fragment can be prepared
as described in the Subheading 3.2.4 (see Fig. 6). Notably, this
newly innovated iRAT segment has a moonlighting function as a
fluorescent tag (see Note 5). The nested mCherry facilitates estimation of the expression levels of antibody fragments by simply
applying the crude culture supernatant to fluorescence sizeexclusion chromatography (FSEC).
iRAT-mediated Fab production can be performed using either
Brevibacillus or Sf9 insect cells (see Note 6). When Sf9 expression is
adopted, the culture supernatant can be directly loaded onto a
column for IMAC using Ni Sepharose excel resin, which saves
time and labor for pretreatment, such as ammonium sulfate precipitation and dialysis.
3.2.1 Construction
of the Baculovirus Strain
The pFBgp67-iRATmC vector (Fig. 4a) for the expression of Fab
fragments is designed based on the backbone of the pFastBac1, an
entry vector to produce Bac-to-Bac baculoviruses. The Fab L insertion site, iRATmC cassette, and Fab H insertion site are tandemly
arranged in this order and are introduced downstream of the constitutively active P PH promoter and gp67 secretion signal sequence.
The iRATmC cassette encodes the N-terminal TEV cleavage site,
His 6 tag, a 35-residue Gly/Ser/Thr-rich linker, the mCherry coding sequence (residues 3–236 from UniProt ID: X5DSL3), a
35-residue Gly/Ser-rich linker, and a C-terminal TEV cleavage
site (Fig. 4c).
1. Day 1: Perform PCR amplifications of the Fab L , iRATmC,
Fab H , and pFastBac vector backbone containing the gp67
signal sequence (VEC-FB). Add the appropriate components
to four 0.2-mL PCR tubes as in Table 5. The PCR primertemplate combinations are listed in Table 6. The primers for
Fab L and Fab H domains include 5
0 overhangs (27 bp) complementing the upstream and downstream sequences to the
pFBgp67-iRATmC vector junctions. Mix the contents gently,
and place the tubes in a thermal cycler.
2. Run the PCR according to the following parameters: (1) 2 min
at 95
C; (2) 30 cycles of a sequence composed of 10 s at
98
C/5 s at 56
C/5 s/kbp at 72
C; and (3) 10 min at 72
C.
3. Check the PCR products by loading an aliquot (5 μL) on a 1%
agarose gel with a DNA ladder in a separate lane. After ethidium bromide staining, observe the gel by UV illumination.
4. Purify the PCR product (45 μL) using a Wizard SV gel and
PCR clean-up system, according to the manufacturer’s instructions. Use the PCR products directly for purification. DNA
extraction from the agarose gel slice is not necessary.
Production of Recombinant Antibody Fragments via the iRAT system
91
removal of the iRAT segment as described in the Subheading
3.2.3 (see Fig. 5). Once the iRAT segment is excised by TEV
protease, a large amount of tag-free Fab fragment can be prepared
as described in the Subheading 3.2.4 (see Fig. 6). Notably, this
newly innovated iRAT segment has a moonlighting function as a
fluorescent tag (see Note 5). The nested mCherry facilitates estimation of the expression levels of antibody fragments by simply
applying the crude culture supernatant to fluorescence sizeexclusion chromatography (FSEC).
iRAT-mediated Fab production can be performed using either
Brevibacillus or Sf9 insect cells (see Note 6). When Sf9 expression is
adopted, the culture supernatant can be directly loaded onto a
column for IMAC using Ni Sepharose excel resin, which saves
time and labor for pretreatment, such as ammonium sulfate precipitation and dialysis.
3.2.1 Construction
of the Baculovirus Strain
The pFBgp67-iRATmC vector (Fig. 4a) for the expression of Fab
fragments is designed based on the backbone of the pFastBac1, an
entry vector to produce Bac-to-Bac baculoviruses. The Fab L insertion site, iRATmC cassette, and Fab H insertion site are tandemly
arranged in this order and are introduced downstream of the constitutively active P PH promoter and gp67 secretion signal sequence.
The iRATmC cassette encodes the N-terminal TEV cleavage site,
His 6 tag, a 35-residue Gly/Ser/Thr-rich linker, the mCherry coding sequence (residues 3–236 from UniProt ID: X5DSL3), a
35-residue Gly/Ser-rich linker, and a C-terminal TEV cleavage
site (Fig. 4c).
1. Day 1: Perform PCR amplifications of the Fab L , iRATmC,
Fab H , and pFastBac vector backbone containing the gp67
signal sequence (VEC-FB). Add the appropriate components
to four 0.2-mL PCR tubes as in Table 5. The PCR primertemplate combinations are listed in Table 6. The primers for
Fab L and Fab H domains include 5
0 overhangs (27 bp) complementing the upstream and downstream sequences to the
pFBgp67-iRATmC vector junctions. Mix the contents gently,
and place the tubes in a thermal cycler.
2. Run the PCR according to the following parameters: (1) 2 min
at 95
C; (2) 30 cycles of a sequence composed of 10 s at
98
C/5 s at 56
C/5 s/kbp at 72
C; and (3) 10 min at 72
C.
3. Check the PCR products by loading an aliquot (5 μL) on a 1%
agarose gel with a DNA ladder in a separate lane. After ethidium bromide staining, observe the gel by UV illumination.
4. Purify the PCR product (45 μL) using a Wizard SV gel and
PCR clean-up system, according to the manufacturer’s instructions. Use the PCR products directly for purification. DNA
extraction from the agarose gel slice is not necessary.
Production of Recombinant Antibody Fragments via the iRAT system
91