Protein Expression and Purification 208–209 (2023) 106289

Contents lists available at ScienceDirectProtein Expression and Purification
journal homepage: www.elsevier.com/locate/yprep

Recombinant production of antibody antigen-binding fragments with an
N-terminal human growth hormone tag in mammalian cells
Yuriko Adachi a, Mika K. Kaneko b, Yukinari Kato b, c, Terukazu Nogi a, *aGraduate School of Medical Life Science, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai, Miyagi, 980-8575, Japanc
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Sendai, Miyagi, 980-8575, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Monoclonal antibody
Fab production
Mammalian cell expression
Human growth hormone
Immobilized metal chelate affinity
chromatography

Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life
science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein
production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an
N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified withstandard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using
the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by
TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at~4 μg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its
native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse
IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method canprovide greater flexibility in functional and structural modifications of target Fabs because specialized purifi­
cation techniques are not necessary.

1. Introduction
Antigen-binding fragments (Fabs) of monoclonal antibodies are
beneficial in both medical and pharmaceutical sciences and in otherfields such as molecular and cellular biology. Notably, Fabs have facil­
itated structure determination of challenging targets in structural
biology. For instance, Fabs serve as crystallization chaperones that
promote packing formation of membrane proteins covered with fluidic
detergent-micelles in X-ray crystallography [1,2]. Fabs have also beenshown
toenablesingle-particle
cryo-EM
analysisoflow-molecular-weight proteins as fiducial markers for particle micro­
graph alignment [3,4]. However, applying Fabs to crystallization andcryo-EM analyses demands their production in milligram or
sub-milligram quantities. There have been two major strategies
employed in the preparation of Fabs: digestion of full-length antibodies
and recombinant production of partial fragments [5]. Fabs arecomposed of the variable heavy (VH), constant heavy 1 (CH1), variable
light (VL), and constant light (CL) regions, and can be prepared bycleaving the hinge region between CH1 and constant heavy 2 (CH2) withpapain. However, the digestion of IgG or IgM requires optimized

reaction conditions and the separation of the post-cleavage fragments,
frequently leading to lower yields. It is also possible to produce Fabs
recombinantly using cDNA in both bacterial and mammalian expression
systems, but the production levels in both systems will vary dependingon the Fab sequence. Additionally, specialized techniques such as af­
finity purification with Protein L-immobilized resin are necessary toseparate Fabs from crude extracts because Fabs lack the Fc region and do
not bind Protein A. Further, the application of Protein L-immobilized
resin is limited to the antibodies containing specific subtypes of the
kappa light chain [6]. Antigen-conjugated resins can be used to purify
Fabs, but these are not generalizable and not commercially available inmost cases.
Thus, we explored an alternative method to prepare Fabs repro­
ducibly by using conventional separation techniques. Specifically, we
produced Fabs as secreted fusion proteins with N-terminal human
growth hormone (hGH) and octa-histidine (His8) tags using a pSGHV0
vector [7] in mammalian cells and to purified them by immobilized
metal chelate affinity chromatography. In this expression system, thehGH and His8 tags can be cleaved off specifically and efficiently by TEVprotease after purification, leaving only a few residues derived from the* Corresponding author.
E-mail address: nogi@yokohama-cu.ac.jp (T. Nogi).
https://doi.org/10.1016/j.pep.2023.106289
Received 11 April 2023; Received in revised form 24 April 2023; Accepted 27 April 2023
Available online 7 May 2023
1046-5928/© 2023 Elsevier Inc. All rights reserved.

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Protein Expression and Purification 208–209 (2023) 106289

Fig. 1. Production of the NZ-1Fab as an hGH-fusion protein (A) Construct design. The hGH and His8 tags and the TEV protease consensus sequence were fused to the
N-terminus of the NZ-1 VH-CH1 region comprised of heavy chain residues E20 to C238. In NZ-1 (rat IgG2a), C238 forms a disulfide bond with the Cys residue at the Cterminus of the light chain. The TEV protease consensus sequence was directly connected to E20 on the VH region in the initial construct while the Ser and Ser-Gly
residues were inserted after the Gly residue of the consensus sequence in the modified constructs. (B, C) Small-scale production of the hGH-fused NZ-1 Fab with noinsertion. The hGH-fused NZ-1 Fabs purified from the culture supernatant with Ni-NTA agarose were separated by SDS-PAGE under reducing (B) and non-reducing(C) conditions. The plasmids encoding the hGH-VH-CH1 and VL-CL regions were mixed at mass ratio indicated above the gel image.

recognition sequence at the N-terminus. We first produced an Fab for the
rat monoclonal antibody NZ-1 (IgG2a, lambda) [8] to demonstrate thefeasibility of the hGH-fusion strategy. The NZ-1 antibody has been uti­
lized in a variety of applications ranging from medical science tobiochemistry. NZ-1 recognizes 14 residues of human podoplanin (PA14)
expressed on cancer cells, and it suppress the platelet aggregation by
inhibiting the binding of CLEC-2 to podoplanin [9]. As NZ-1 shows an
extremely high affinity to the 12 C-terminal residues (PA12) of PA14,
the PA12/NZ-1 pair has been developed as an immunoaffinity purifi­
cation system [10]. Furthermore, NZ-1 forms stable complexes with
PA12- or PA14-grafted target proteins [11,12], raising possibilities that
the NZ-1 Fab can serve as crystallization chaperone or fiducial markers
in structural analysis. After examining the structure and function of theNZ-1 Fab produced as the hGH-fusion protein, we also demonstrated thebroad applicability of our hGH-fusion strategy by producing several
mouse monoclonal antibodies belonging to IgG1, the major subclass formouse IgG.

ratio of 10:1 in the transfection (Fig. 1B and C). Subsequently, we
attempted to remove the hGH-His8 portion by adding TEV protease to
the elution fraction. However, majority of hGH-VH-CH1 remained
uncleaved (Fig. 2A, D). In this construct, the TEV protease consensus
sequence was directly fused to the N-terminal residue of VH (Glu-20)(Fig. 1A). Thus, we inferred that the steric hindrance between TEVprotease and VH reduced the cleavage efficiency.
2.2. Optimization of TEV cleavable linker for efficient removal of the Nterminal tags
We next modified the fusion protein to eliminate steric hindrance for
TEV protease. TEV protease cleaves between Gln and Gly in theconsensus sequence (ENLYFQG). We inserted linker residues, Ser or SerGly, after the Gly residue in the consensus sequence (Fig. 1A). The
cleavage efficiency was greatly improved for the two resulting insertion
mutations such that the vast majority of both were cleaved by TEVprotease (Fig. 2B,C,E,F) under the previous reaction conditions. We
therefore worked to establish a purification protocol for Fabs using theconstruct with the more minimal single Ser insertion (hereafter, thehGH-fused Fab is referred to as hGH-GS-Fab and the post-cleavage
product is referred to as GS-Fab). After the tag cleavage, hGH-His8 andHis-tagged TEV protease were removed using Ni-NTA agarose resin.
Finally, gel filtration was performed to collect the GS-Fab in a mono­disperse state (Fig. 3A and B).

2. Results and discussion
2.1. Production of hGH-fused fab
We produced the heavy chain fragment as a hGH-fusion protein(Fig. 1A), which mediates secretion of the recombinant fusion protein.
The cDNA encoding residues 20 to 238 of the NZ-1 heavy chain (cor­responds to the VH-CH1 regions) was cloned into the pSGHV0 secretion
expression vector. The constructed plasmid was then transfected into
mammalian cells (Expi293F) together with a plasmid encoding thecDNA of the NZ-1 light chain. When the culture supernatant was applied
to Ni-NTA agarose, a disulfide-linked complex of the hGH-VH-CH1 and
VL-CL segments appeared in the elution fraction as confirmed by SDSPAGE. Furthermore, the expression level of the complex increased
when the plasmids for the heavy and light chains were mixed at a mass

2.3. Flow cytometry to examine the binding affinity to cell surface
antigens
We subsequently examined if the recombinant NZ-1 GS-Fab main­tained its function. NZ-1 was originally established as a highly reactive
antibody against human podoplanin and was used to suppress
podoplanin-induced platelet aggregation. It has also been reported that2

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Protein Expression and Purification 208–209 (2023) 106289

Fig. 2. Cleavage of the N-terminal tags by TEV protease. (A, D) The initial construct with no insertion. (B, E) The modified construct with the single Ser insertion (NZ1 GS-Fab). (C, F) The modified construct with the Ser-Gly insertion. The hGH-fused NZ-1 Fab samples before (-) and after (+) cleavage were separated by SDS-PAGE
under reducing (A–C) and non-reducing (D–F) conditions. The bands for the hGH and the VH-CH1 fragments generated by the cleavage appeared to overlap under
reducing conditions.

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Protein Expression and Purification 208–209 (2023) 106289

Fig. 3. Analytical gel filtration of the NZ-1 GS-Fab.
(A) The monodisperse peak of the NZ-1 GS-Fab
appeared in the elution from gel filtration. The six
fractions around the peak (grey background) wereseparated by SDS-PAGE under reducing conditions in(B). (C) When mixed with PDZ tandem(181-PA14184), the peak shifted to the higher molecularweight side while a peak of residual PDZ tandem wasalso observed. The six fractions containing the com­plex and the residual PDZ tandem (grey background)
were separated by SDS-PAGE under reducing condi­
tions in (D).

NZ-1 strongly bound with human podoplanin expressed on CHO cells.
We therefore performed flow cytometry on podoplanin-expressing CHO(CHO/hPDPN) cells to compare the binding affinities between the NZ-1
GS-Fab and the IgG-digested NZ-1 Fab. We applied serial two-folddilution of GS-Fab or IgG-digested Fab ranging from 10 to 0.0006 μg/
mL, which corresponds to 240 to 0.013 nM, to the CHO/hPDPN cells.
Binding data were fit to a sigmoidal 2-state binding model, and thedissociation constants for GS-Fab and IgG-digested Fab were estimatedat 2.37 and 5.19 nM, respectively (Fig. 4). It is therefore presumed thatthe complementarity determining regions were properly formed in the
NZ-1 GS-Fab produced as a hGH-fusion protein despite the difference of
expression method and the presence of the additional Gly-Ser residues at
the N-terminus as compared to the IgG-digested NZ-1 Fab.

Considering that the recovery of Fab from IgG after the papain digestionis only about 20% in some cases, the yield of NZ-1 GS-Fab (~4 μg/mL ofculture supernatant) would be comparable to that of IgG-digested NZ-1
Fab.
NZ-1 GS-Fab complexed with PDZ tandem(181-PA14-184) produced
crystals that diffracted X-ray to 2.2 Å resolution. The crystal structure
was solved by molecular replacement method using the atomic co­ordinates of the IgG-digested NZ-1 Fab complexed with PDZ tandem(181-PA14-814) as a search model (Table 1). The atomic models were
almost identical between the two complexes where the 426 Cα atoms in
the Fabs were superposed onto one another with RMSD = 0.247 Å(Fig. 5A–C). The Gly-Ser residues left on the N-terminus of the heavy
chain after tag cleavage were far removed from the antigen-binding
pocket and not involved in the interaction with PDZ tandem(181PA14-184). The second Ser residue was clearly observed in the elec­tron density map while the model for the first Gly residue was not
assigned in the crystal structure due to disorder (Fig. 5D). These results
confirmed that the NZ-1 GS-Fab prepared from the hGH-fusion protein
adopted the quaternary structure equivalent to the Fab derived from theintact IgG.

2.4. Structural analysis to verify the folding of GS-Fab
To test if the entire NZ-1 GS-Fab maintains its native folding anddisulfide bonding pattern, we also performed X-ray crystallographic
analysis on the complex with a PA14-grafted protein. In a previous
study, we demonstrated that the PA14 sequence can be inserted intoβ-hairpin regions in the periplasmic domain of an intramembrane pro­tease, the PDZ tandem fragment of the RseP orthologue from the hy­
perthermophile Aquifex aeolicus. We also crystallized the complex of the
IgG-digested NZ-1 Fab and the PA14-grafted protease extracellulardomain, termed PDZ tandem(181-PA14-184), in which PA14 is inserted
between Arg-181 and Glu-184 [12]. In this study, we performed
analytical gel filtration to examine if the above purified NZ-1 GS-Fab
forms a stable complex with PDZ tandem(181-PA14-184). The elution
peak for NZ-1 GS-Fab was indeed shifted to the higher molecular weightside when mixed with PDZ tandem(181-PA14-184). The complex eluted
in a monodisperse state with no apparent dissociation, indicating that
GS-Fab was tightly bound to the PA14-grafted PDZ tandem (Fig. 3C and
D). Subsequently, we purified approximately 1 mg of NZ-1 GS-Fab froma 240 mL culture of Expi293F cells to prepare the complex in larger
quantities. Yields of IgGs prepared from the hybridoma vary widely, but
typically fall in the range of 10–100 μg/mL of culture supernatant.

2.5. Examination of the generalizability of the hGH-fusion method
Lastly, we attempted to overproduce Fabs that differ from NZ-1, a rat
IgG2a with a lambda light chain. We selected Fabs from mouse IgG1s
with kappa light chains: LpMab-7 against human podoplanin, 8B8against the PDZ tandem fragment of RseP orthologue from A. aeolicus,
13F1 against the PDZ tandem fragment of RseP from E. coli. As with the
NZ-1 GS-Fab, the hGH-His8 tag was fused to the N-terminus of the heavy
chain with the single Ser insertion between the TEV protease consensus
sequence and the VH region for each of these four Fabs. All tested Fabconstructs were successfully produced in mammalian cells, and as
anticipated, the tags were digested almost completely after affinity pu­
rification (Fig. 6) without additional protocol optimization. These re­sults support the applicability of the hGH-fusion method constructed inthis study.

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Table 1
Statistics for X-ray crystallographic analysis.
Data Collection
Space group
Cell dimensions
a, b, c (Å)α, β, γ (◦ )No. of complex/a.s.u.
X-ray source
Wavelength (Å)
Resolution limits (Å)
No. of unique reflection
Completeness (%)
Redundancy<I/σ(I)>
RmergeaCC (1/2)

P21212151.94, 75.09, 173.52
90, 90, 90
1PF/BL-17A
0.980045.82–2.20 (2.27–2.20)
35,390 (3,029)
100 (100)
6.7 (6.9)
16.7 (1.7)
0.076 (1.205)
0.999 (0.738)

Refinement
Resolution limits (Å)
RworkbRfreecNo. of non-H atoms
Average B-factor (Å2)
RMSD from ideality
Bond length (Å)/angle (◦ )Ramachandran plot
Favored/Outlier (%)
PDB code

38.32–2.20 (2.23–2.20)
0.2427 (0.3500)
0.2698 (0.3562)
4,802
62.38
0.002/0.54
95.51/0.50
8IPC

Values in parentheses are for highest-resolution shell.
aRmerge = ΣhΣi|Ii(h)-<I(h)>|/ΣhΣiI(h), where Ii (h) is the ith measurement.
b
Rwork is the crystallographic R-factor (Rcryst) for the working set used for therefinement. Rcryst = Σh||Fobs(h)|-|Fcalc(h)||/Σh| Fobs(h)|, where Fobs(h) and Fcalc(h)are the observed and calculated structure factors.
cRfree is Rcryst calculated for the test set consisting of 5% of reflections
excluded from the refinement.

unrelated mouse monoclonal antibodies without the need for additional
protocol optimization demonstrates the generalizability of this method.
Because specialized purification techniques and re-optimized tag
cleavage conditions were not necessary, this method will provide
greater flexibility in functional and structural modifications of target
Fabs.

Fig. 4. Estimation of binding affinities of NZ-1 Fabs by flow cytometry
CHO/hPDPN cells were resuspended at 100 μl of serially diluted NZ-1 GS-Fabproduced as the hGH-fusion protein in (A) or NZ-1 Fab prepared by the IgG
digestion in (B). KD was calculated by fitting the binding isotherms of geometricmean of fluorescence intensities.

CRediT author statement
Yuriko Adachi: Investigation, Writing – original draft.
Mika K. Kaneko: Investigation.
Yukinari Kato: Investigation, Funding acquisition, Supervision,
Writing – original draft, Writing – review & editing.
Terukazu Nogi: Conceptualization, Investigation, Funding acquisi­
tion, Project administration, Writing – original draft, Writing – review &editing.

3. Conclusion
In this study, we have established an overproduction method for Fabs
as hGH-fusion proteins. This method simplifies Fab purification,
replacing specialized affinity resins with resins for conventional metalchelating affinity chromatography. The only structural difference be­
tween the Fabs prepared from the hGH-fusion and from the papain
digestion of intact antibodies is the presence and absence of two residues(Gly-Ser) at the N-terminus of the heavy chain, which are expected tohave little impact on the functions such as antigen recognition andneutralizing activity. Consistent with this expectation, the NZ-1 Fabprepared by the hGH-fusion was successfully crystallized in complex
with an epitope-grated protein while it maintained its binding affinity
for cell-surface antigens. Successful overproduction of Fabs from

Declaration of competing interest
The corresponding author declares no financial and non-financial
competing interests on behalf of all authors.
Data availability
Data will be made available on request.

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Fig. 5. Crystallographic analysis of the NZ-1 GS-Fab.
(A) Crystal structure of the NZ-1 GS-Fab in complex
with PDZ tandem(181-PA14-184). The grafted PA14
epitope was colored blue. The Ser residue inserted
between the TEV protease consensus sequence andthe VH region was depicted with spheres and labeled
S19. (B) Crystal structure of the IgG-digested NZ-1
Fab in complex with PDZ tandem(181-PA14-184).
The previously determined structure (PDB code:
7CQC) is shown in the same view as in (A). (C) Su­
perposition of the two complexes. The Cα traces of thetwo complexes are shown in the same colors as in (A)and (B). (D) Omit map around the N-terminus of theheavy chain. The mFo-DFc map (green mesh) was
calculated by omitting the atomic coordinates of theresidues S19-Q22 from the final model. Atomic co­ordinates S19 could be fit to the map, but no obvious
electron density was observed for Gly18. The putative
position of Gly18 is indicated by a magenta dotted
circle.

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Protein Expression and Purification 208–209 (2023) 106289

Fig. 6. Production of Fabs from mouse IgG1s as hGH-fusion proteins (A, D) LpMab-7. (B, E) 8B8. (C, F) 13F1. The hGH-fused mouse IgG1 Fab samples before (-) andafter (+) cleavage were separated by SDS-PAGE under reducing (A–C) and non-reducing (D–F) conditions.

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Protein Expression and Purification 208–209 (2023) 106289

Acknowledgements

equilibrated in 10 mM Tris-Cl (pH 7.4), and 150 mM NaCl. The peakfractions containing target proteins were concentrated by ultrafiltration
using an Amicon® Ultra Centrifugal Filter with a molecular weight cutoff of 30 kDa (Merck Millipore).

We are grateful to the beamline staff of Photon Factory (Tsukuba,
Japan) for providing data collection facilities and support. We thankSamuel Thomson for editing the manuscript, and associate profs. Nor­
imichi Nomura and Satoshi Ogasawara, and prof. Taro Tamada forvaluable discussions and information on Fab preparation. This research
was partially supported by the Japan Society for the Promotion of Sci­
ence (JSPS) KAKENHI under Grant Numbers JP22H02561, andJP19H03170 (to T.N.), by Japan Agency for Medical Research andDevelopment (AMED) under Grant Number: JP22ama121008 (to Y.K.),
by the Cooperative Research Program (Joint Usage/Research Centerprogram) of Institute for Life and Medical Sciences, Kyoto University (toT.N.), and by the Cooperative Research Project Program of Life Science
Center for Survival Dynamics, Tsukuba Advanced Research Alliance(TARA Center), University of Tsukuba (to T.N.). This work was per­formed partially under the Collaborative Research Program of Institutefor Protein Research, Osaka University, CR-21-01.

Determination of binding affinities by flow cytometry
Serially diluted antibodies (10 μg/ml - 0.6 ng/ml) were treated with
CHO/hPDPN cells. The cells were further incubated with Alexa Fluor
488-conjugated anti-rat IgG (1:200; Cell Signaling Technology, Inc.,
Danvers, MA, USA). Fluorescence data were collected using a BD FAC­
SLyric flow cytometer and analyzed using BD FACSuite software version
1.3 (BD Biosciences, Franklin Lakes, NJ, USA). The KD values were
determined by GraphPad Prism 8 (the fitting binding isotherms to builtin one-site binding models; GraphPad Software, Inc., La Jolla, CA, USA).
X-ray crystallographic analysis
The PA14-grafted PDZ tandem fragment, PDZ tandem(181-PA14184), was produced as an N-terminal GST-fusion protein in E. coli
BL21(DE3) cells. The purification procedure was essentially the same asreported previously [12]. The purified PDZ tandem(181-PA14-184) was
mixed with NZ-1 GS-Fab and applied to a Superdex™ 200 Increase
10/300 GL SEC column (Cytiva) equilibrated with 10 mM Tris-Cl (pH7.4), and 150 mM NaCl to fractionate the complex.
Initial crystallization conditions were searched using the Index™
(Hampton Research) screening kit. 0.2 μL each of protein solution andcrystallization buffer were dispensed into 96-well plates using a Crystal
Gryphon (Art Robbins Instruments) and equilibrated against 60 μL of
crystallization buffer in the reservoir by the sitting-drop vapor-diffusion
method. After optimization, PDZ tandem(181-PA14-184) complexedwith the NZ-1 GS-Fab was crystallized in a solution containing 14%(wt./vol.) polyethylene glycol 3,350, 0.2 M lithium sulfate, and 0.1 M
Bis-Tris-HCl (pH 6.5).
Crystals were flash frozen in liquid nitrogen after being quickly
soaked in cryoprotectant, which was prepared by mixing the crystalli­zation buffer and ethylene glycol in a ratio of 4:1 (vol/vol). X-ray
diffraction data were collected using PILATUS3 S 6 M (Dectris) at
Photon Factory (PF) BL-17A (Tsukuba, Japan). Data were processed andscaled with XDS [16] and aimless [17]. Diffraction intensities were
subsequently converted to structure factors using the CCP4 suite [18]where 5% of the unique reflections were randomly selected for the
calculation of free R-factor.
Initial phases were determined by the molecular replacement
method using Molrep [19], in which the crystal structure of the PDZ
tandem(181-PA14-184) complexed with IgG-digested NZ-1 Fab wasused as a search model. The initial model was manually modified and fit
into the electron density map using the program COOT [20]. The
updated models were refined with phenix.refine [21] iteratively while
monitoring the stereochemistry with MolProbity [22]. Statistics for
X-ray crystallographic analysis are summarized in Table 1. Structural
superposition and RMSD calculation were performed by a pair-wisealignment protocol using LSQKAB [23]. Figures for protein structures
were prepared with PyMOL (The PyMOL Molecular Graphics System,
Version 2.3 Schrödinger, LLC.).
The atomic coordinates and structure factors of PDZ tandem(181PA14-184) complexed with NZ-1 GS-Fab have been deposited in theProtein Data Bank with an accession number of 8IPC.

Material and Methods
Antibodies and expression plasmids
NZ-1 was generated by immunizing rats with 14 amino acids fromresidue 38 to residue 51 of human podoplanin [8]. LpMab-7 wasgenerated by immunizing mice with human podoplanin-overexpressed
LN229 glioblastoma cells [13]. 8B8 and 13F1 were generated byimmunizing GANP mice [14] with a mixture of PDZ tandem fragmentsof E. coli RseP and its orthologue from Aquifex aeolicus as reported
previously [15]. 8B8 was selected as a monoclonal antibody against the
PDZ tandem from A. aeolicus RseP while 13F1 was selected against the
PDZ tandem from E. coli RseP. For the heavy chain of NZ-1, cDNA for the
VH-CH1 region was subcloned between the BamHI and EcoRI sites in thepSGHV0 vector using the In-Fusion HD Cloning Kit (Takara Bio). The Seror Ser-Gly insertions between the TEV protease consensus sequence andthe NZ-1 VH region were introduced by the inverse PCR method. For theheavy chains of LpMab-7, 8B8, and 13F1, cDNAs encoding the VH-CH1
region were amplified with primers that added an additional Ser residue
at the N-terminus. These cDNAs were then subcloned into the pSGHV0
vector [7]. The cDNAs encoding the light chains with the original signal
sequences were subcloned into the pCAG-Neo vector (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan).
Protein expression and purification
Expression plasmids for the heavy and light chains were mixed at
mass ratios of 10:1, 1:1, or 1:10 and transfected into Expi293F™ cells(Thermo Fisher Scientific) using the ExpiFectamine™ 293 Transfection
Kit (Thermo Fisher Scientific). The cells were cultured in 2 mL medium
in a 6-well plate, or 30 mL medium in a 125-mL flask. Larger production
batches used multiple 30 mL cultures. After pelleting the cells by
centrifugation, the culture supernatant was neutralized by adding TrisCl (pH8.0) at a final concentration of 20 mM for efficient target pro­tein binding to Ni-NTA agarose resin (QIAGEN). After incubating withthe culture supernatant, the resin was washed with 20 mM Tris-Cl (pH8.0), 300 mM NaCl, and 10 mM imidazole. Subsequently, the captured
proteins were eluted with 20 mM Tris-Cl (pH8.0), 300 mM NaCl, and
250 mM imidazole. N-terminally hexahistidine-tagged TEV protease(His6-TEV) was added to the elution fraction to cleave off the N-terminal
hGH and His8 tags. For further purification, the cleavage fraction was
dialyzed against 10 mM Tris-Cl (pH 8.0), and 150 mM NaCl overnight at
4 ◦ C using the dialysis membrane with molecular weight cut-off of
3,500. The cleaved hGH-His8 portion and His6-TEV were removed bypassing the fraction through Ni-NTA agarose resin. Finally, the flowthrough and wash fractions containing Fabs were further purified bygel filtration on a Superdex™ 200 Increase 10/300 GL (Cytiva) column

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