Balanced expression of single subunits in a multisubunit protein,
achieved by cell fusion of individual transfectants
Lars Norderhaug
1
, Finn-Eirik Johansen
2
and Inger Sandlie
3
1
Antibody Design AS, Nesoddtangen, Norway;
2
Department of Pathology, Rikshospitalet, Norway;
3
Department of Biology,
University of Oslo, Norway
To establish stable cell lines that produce recombinant
multisubunit proteins, it is usually necessary to cotransfect
cells with several independent gene constructs. Here, we
show that a stepwise fusion of individually transfected cells,
results in a fused cell-line that secretes a complete multi-
subunit protein. Functional expression of recombinant
multisubunit proteins may require a defined expression ratio
between each protein subunit. The cell-fusion technology
described allows a predefined expression level of each sub-
unit. Using SIgA as a model protein we demonstrate that the
majority of the fused cells inherit the molar expression ratio
of the parental transfected cells. These results indicate that
simplified screening of clones expressing the expected sub-
unit ratios may be possible using the cell-fusion technology.
This technology may therefore be an alternative to generic
transfection methods for the establishment of cells that

the development and application of this technology for
production of recombinant multisubunit proteins has not
been described previously. Here, we achieve multigene
expression by utilizing cell-fusion of individually transfected
cells, each expressing one or more genes that encode the
multisubunit protein. We show that the majority of clones
resulting from the fusion inherit the expression levels of the
parental cells, thus simplifying screening for clones with
stochiometric expression levels of each component that
secrete fully functional SIgA. Furthermore, we show that this
system enables high-level expression in mammalian cells,
which is often a goal in recombinant protein expression.
MATERIAL AND METHODS
Vectors and cloning of genes
Construction of the vector family pLNO and its use for
transfection and expression of immunoglobulin genes has
been described previously [20–22]. The human Ig a1
gene was subcloned into pLNO/Neo giving the vector
pLNOA1/Neo. The Ig heavy-chain variable-gene (VH)
SS-269VH [23], specific for the outer membrane protein of
the bacteria Neisseria meningitides, was subcloned into
pLNOA1/Neo giving pLNOA1/Neo-SS269. The human
Ig k gene was subcloned into pLNO/Neo, giving pLNOL/
Neo. The Ig light-chain variable-gene (VL) SS-269VL [23],
specific for the outer membrane protein of the bacteria
N. meningitides, was subcloned into pLNOL/Neo giving
pLNOL/Neo-SS269. The construction of the human
J-chain vector pCH (CMV-driven expression, hygromy-
cin B resistance) [2] and the human SC vector pcDNA(zeo)-
His

at 0 °C giving a 12-ms pulse.
Following transfection, the cells were subsequently trans-
ferred into 25 mL Dulbecco’s modified Eagle’s medium
(DMEM) or HAM F-12 with 10% fetal bovine serum in
25 cm
2
flasks,andallowedtorecoverfor24hbefore
addition of appropriate antibiotics; 800 lgÆmL
)1
G418
(Invitrogen BV, the Netherlands) or 400 lgÆmL
)1
hygromy-
cin (Invitrogen BV, the Netherlands) or 400 lgÆmL
)1
Zeocin
(Invitrogen BV, the Netherlands). Three individual transfec-
tions were employed: (a) the vector pCH/J-chain/Hygro (J-
chain vector) (b) the vector pcDNA (zeo)his6/SC (SC vector)
and (c) cotransfection of the vectors pLNOL/Neo-SS269 (k
vector) and pLNOA1/Neo-SS269 (a1vector)inCHO-K1
cells. Cells were allowed to grow for 10 days before protein
expression was analysed.
Cell fusion
Cell fusion was performed in two individual steps (Fig. 1) by
mixing equal number of cells (3 · 10
7
cells) of each fusion
partner. Cells were centrifuged (5–10 min, 200–400 g) and
washed once in serum free medium. The cell mixture was

plates coated with 4 lgÆmL
)1
of N. meningitides OMV
(a gift from T. E. Michaelsen, National Institute of Public
Health, Norway). Secondary antibodies used for detection
were rabbit anti-(human IgA) Ig (DAKO; 1 : 5000 dilution)
and rabbit anti-SC Ig (DAKO; 1 : 3000 dilution) and
tertiary antibody used for detection was horseradish
peroxidase (HRP)-conjugated goat anti-(rabbit IgG) Ig
(DAKO; 1 : 3000 dilution). The absorbance was read by
TitertekÒ Multiskan (ICN Flow, USA). The amount of
IgA present in each supernatant was calculated relative to a
standard preparation with known concentration.
Verification of J-chain expression
To examine production of J-chain, transfected cells were
screened by immunofluorescence staining. CHO-K1 cells
transfected with J-chain were cultured on micro slides. Cells
were fixed and permeabilized in methanol ()20 °Cfor
4 min). The cells were then washed twice with NaCl/P
i
and
incubated at room temperature for 20 min with 1 : 3000
diluted rabbit anti-(J-chain) Ig (P. Brandtzaeg, LIIPAT,
National Hospital, Norway). Cells were then washed three
times with NaCl/P
i
and incubated for 20 min with (1 : 200)
fluorescein isothiocyanate (FITC)-conjugated goat anti-
(rabbit IgG) Ig (DAKO). Cells were washed as above and
analysed by fluorescence microscopy.

NaCl/P
i
/Tween before addition of substrate (Bio-Rad) for
5 min. The membranes were covered with plastic film and
exposed to Kodak X-OMAT film for 15–60 s. Dot blot
density was analysed by
TOTALLAB
gel software (Phonetix,
UK). The amount of SC present in each supernatant was
calculated relative to a standard preparation with known
concentration.
Western blot of IgA, pIgA and SIgA
Aliquots of 10 lL supernatant from selected clones were
analysed under nonreducing conditions on a 4–15% Tris/
HCl SDS/PAGE ReadyGel (Bio-Rad) run at 200 V for 1 h.
The gel was blotted onto PVDF paper (Millipore; Sweden)
in a Bio-Rad Miniblotter for 1 h at 100 V. Following the
transfer, the membranes were washed in NaCl/P
i
for
5–10 min with gentle agitation, and blocked for 45 min in
NaCl/P
i
/Tween with 10% skimmed milk. The membrane
was washed once in NaCl/P
i
before incubation with either
1 : 5000 dilution rabbit anti-(human IgA) or 1 : 3000
dilution rabbit anti-(human SC) Ig for 1 h. The membranes
were washed twice followed by incubation with 1 : 3000

J-chain. A further attempt to directly quantify the amount
of intracellular J-chain was avoided, as retention is closely
linked to degradation [24,25]. One clone, J-1, with high
fluorescence intensity was selected for further expansion and
fusion to IgA-29. The SC gene was transfected into CHO-
K1 cells on the vector pcDNA(zeo)his6/SC. Twenty-four
clones were analysed by dot blot as described, and six of
these were positive for SC production. One clone, SC-4,
was expanded for further fusion. The clones IgA-29
(4.5 lgÆmL
)1
) and SC-4 (2.3 lgÆmL
)1
) were chosen for cell
fusions because the amount of expressed protein on a molar
basis is almost equal in these cells, as the M
r
of IgA and SC
are 160 and 80 kDa, respectively.
Fusion of single transfectants to achieve
SIgA-producing clones
The first fusion of IgA-producing cells (IgA-29) and J chain-
producing cells (J-1) resulted in numerous G418 and
hygromycin B resistant colonies. The overall fusion effi-
ciency was as high as 1 · 10
)3
. Five colonies were analysed
for production of IgA and J-chain by ELISA. All five
colonies were shown to produce both polypeptide chains.
One clone (pIgA-D) producing polymeric IgA was expan-

for SC (Fig. 2A).
The molar expression ratio between IgA and total SC was
calculated for each fused clone, and compared with the
molar expression ratio of the parental cells SC-4/pIgA-D.
The molar ratio varied from 0.9 to 2.2, while 50% of the
clones maintained the molar ratio of  1 (Fig. 2B). This
shows that the selection and isolation of clones expressing a
stochiometric or predefined ratio of different protein
subunits is well within reach of a simple screening proce-
dure. SC bound to IgA also correlated with IgA expression
levels in all fused clones shown by ELISA (data not shown).
Because SC only interacts with J-chain-positive pIgA, the
Ó FEBS 2002 Multi-subunit protein production by cell fusion (Eur. J. Biochem. 269) 3207
complexing of IgA and J-chain is a prerequisite for SC
binding. Therefore, the correlation between the IgA and SC
levels in all the clones demonstrated that a sufficient amount
of J-chain was available for SIgA complex formation. One
SIgA-producing clone (SIgA-3) along with pIgA-D and
IgA-29 were analysed by SDS/PAGE gel and Western blot
(Fig. 3) to characterize the molecular size and composition
of the secreted products. This Western blot clearly demon-
strated that cells fused to produce all the four polypeptides
of SIgA, assembled and secreted SIgA of the expected
molecular size with reactivity against antibodies towards
both IgA and SC. The fused cells expressed both monomeric
and polymeric IgA as described for hybridoma cells
expressing monoclonal IgA [27], and was also seen when
the J-chain gene was transfected into an IgA-producing
CHO cell line [2]. The production level of the fused cells
grown in culture, without any selective pressure, was

[30] of chromosomal integrating vectors. However, for
stable expression of multiple genes over time, the usefulness
of such episomal vector is limited, mainly because of the
slow loss of the vector over time when unselected [39]. A
defined expression ratio between each protein subunit may
be essential for the specific function of the mature protein
product. Study of ion channels [8] and receptors [3,41] have
shown that their structure and functions actually depend on
the level of expression of the different subunits. Thus, cell-
fusion technology will allow generation of cells with a
Fig. 3. Western blot of assembled IgA, pIgA
and SIgA. SDS/PAGE and Western blot of
untransfected CHO-K1 cells (lane 1), IgA-29
(lane 2), pIgA-D (lane 3 and 5) and SIgA-3
(lane 4 and 6) detected by antihuman IgA or
strippedandredetectedwithantihumanSC,as
indicated. The blot shows assembly of all
protein subunits in the fused cells.
Fig. 2. IgA and total SC production levels (A) and molar expression
ratios (B). (A) IgA and SC expression levels of outgrown supernatants
of the clones: SC-4, IgA-29, pIgA-D and SIgA-1–8 measured in triplets
by ELISA or dot blot. (B) Calculation of the molar expression ratio
between the subcomponents SC and IgA in each fusion clone SIgA1-8
and also between their parental cells SC-4 and IgA-29. Calculations
were based on measured expression levels of both SC and IgA (A) and
the M
r
of SC (80 kDa) and IgA (160 kDa).
3208 L. Norderhaug et al. (Eur. J. Biochem. 269) Ó FEBS 2002
predefined expression level of each protein subunit and

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