FDA-approved therapeutic antibodies. The resulting Fv fragments
are functionally active and are crystallized in complex with the
target proteins [4, 10–12].
Another important feature of the iRAT system is its high extensibility. Antibodies in different modalities can be produced via the
original and modified iRAT system. Beyond the production of Fv
fragments, we have succeeded in producing Fab fragments through
a process of reorganization of the iRAT segment. Interestingly,
when a polyhistidine-mCherry (His 6 -mCherry) cassette is
incorporated within the long intervening region, tethering the
light and heavy chains of Fab fragments, the resultant polyproteins
fold and assemble into functional fluorescent Fab fragments
(Fig. 1b). The iRAT concept could provide promising clues to
design and create antibody-derived artificial molecular devices
that are useful in various basic biomedical and clinical procedures,
such as fluorescence-activated cell sorting (FACS), fluorescencelinked immunosorbent assay (FLISA), immunohistochemistry
(IHC), fluorescent bioimaging, and immunodiagnostics.
In this chapter, we present reliable and broadly applicable iRAT
protocols to prepare milligram quantities of Fv and Fab antibody
fragments using Brevibacillus and Sf9 insect cells, respectively, with
an average duration. Anticipated results are also shown, taking
examples of the iRAT-mediated production of certolizumab Fv
and Fab. Certolizumab (Cimzia
®
) is an FDA-approved humanized
monoclonal antibody that targets tumor necrosis factor α (TNFα)
and is widely used to treat rheumatoid arthritis and Crohn’s disease.
Biosimilars of such known therapeutic antibodies for research use
can be easily prepared via the iRAT system. Both Fv production
using Sf9 cells and Fab production using Brevibacillus can be
performed similarly, although the detailed protocols are not
described here due to space limitations.
2 Materials
2.1 Molecular
Biology
1. PrimeSTAR Max Premix (2Â) (Takara Bio/Clontech).
2. Gibson Assembly Master Mix (2Â) (New England Biolabs).
3. TaKaRa Ex Taq (5 U/μL) (Takara Bio/Clontech).
4. 10Â Ex Taq Buffer.
5. dNTPs mixture (2.5 mM each).
6. Wizard SV gel and PCR clean-up system (Promega).
7. MinElute PCR purification kit (Qiagen).
8. QIAquick spin miniprep kit (Qiagen).
9. Thermal cycler.
10. NanoDrop spectrophotometer.
Production of Recombinant Antibody Fragments via the iRAT system
79
are functionally active and are crystallized in complex with the
target proteins [4, 10–12].
Another important feature of the iRAT system is its high extensibility. Antibodies in different modalities can be produced via the
original and modified iRAT system. Beyond the production of Fv
fragments, we have succeeded in producing Fab fragments through
a process of reorganization of the iRAT segment. Interestingly,
when a polyhistidine-mCherry (His 6 -mCherry) cassette is
incorporated within the long intervening region, tethering the
light and heavy chains of Fab fragments, the resultant polyproteins
fold and assemble into functional fluorescent Fab fragments
(Fig. 1b). The iRAT concept could provide promising clues to
design and create antibody-derived artificial molecular devices
that are useful in various basic biomedical and clinical procedures,
such as fluorescence-activated cell sorting (FACS), fluorescencelinked immunosorbent assay (FLISA), immunohistochemistry
(IHC), fluorescent bioimaging, and immunodiagnostics.
In this chapter, we present reliable and broadly applicable iRAT
protocols to prepare milligram quantities of Fv and Fab antibody
fragments using Brevibacillus and Sf9 insect cells, respectively, with
an average duration. Anticipated results are also shown, taking
examples of the iRAT-mediated production of certolizumab Fv
and Fab. Certolizumab (Cimzia
®
) is an FDA-approved humanized
monoclonal antibody that targets tumor necrosis factor α (TNFα)
and is widely used to treat rheumatoid arthritis and Crohn’s disease.
Biosimilars of such known therapeutic antibodies for research use
can be easily prepared via the iRAT system. Both Fv production
using Sf9 cells and Fab production using Brevibacillus can be
performed similarly, although the detailed protocols are not
described here due to space limitations.
2 Materials
2.1 Molecular
Biology
1. PrimeSTAR Max Premix (2Â) (Takara Bio/Clontech).
2. Gibson Assembly Master Mix (2Â) (New England Biolabs).
3. TaKaRa Ex Taq (5 U/μL) (Takara Bio/Clontech).
4. 10Â Ex Taq Buffer.
5. dNTPs mixture (2.5 mM each).
6. Wizard SV gel and PCR clean-up system (Promega).
7. MinElute PCR purification kit (Qiagen).
8. QIAquick spin miniprep kit (Qiagen).
9. Thermal cycler.
10. NanoDrop spectrophotometer.
Production of Recombinant Antibody Fragments via the iRAT system
79