Acta Biochim Biophys Sin, 2020, 00(00), 1–3
doi: 10.1093/abbs/gmaa055
Lab Note
Production of integrin αIIbβ3 in stably
transfected and clonal Chinese hamster ovary
cells for functional and structural studies
Li Pan1 , Ying Lu2 , Yongmei Zuo3 , Kechang Qu1 , Wenlei Ma1, and
Jiafu Liu1,*
1 College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China, and 2 College of Life
Sciences, Northeast Agricultural University, Harbin 150030, China, and 3 Heilongjiang Institute of Animal Health
Inspection, Harbin 150006, China
∗ Correspondence address. Tel/Fax: +86-451-55190083; E-mail: liujiafu@neau.edu.cn
The essential prelude for structural biologists is to choose the rea-
sonable expression methods, which are categorized into eukaryotic
and prokaryotic expression systems, to obtain sufficient amount
of protein for structure determination [1]. For many human pro-
teins, especially membrane receptors, the prokaryotic system cannot
be amenable for target-protein expression. It is often difficult to
generate properly folded, fully functional eukaryotic proteins in
prokaryotes. A variety of eukaryotic expression systems have been
explored to overcome this problem, including yeast, insect cells,
and mammalian cells [2,3]. The mammalian cell expression system
is popular now for the overexpression of mammalian proteins for
functional and structural studies. For the expression and purification
of heterodimeric membrane proteins, subunit topology and construct
design are the other important aspects. The subunits can be expressed
in different host cells separately and then reconstituted in vitro. They
also can be expressed in the same host cell to form the dimeric
proteins in vivo [4–6].
For heterodimeric or heteromultimeric proteins, co-expression of
subunits with their assembly into a functional entity in the same
hosts has become more and more feasible. Co-expression can be
executed using the single or multiple constructs. When two or more
constructs are co-transformed into a single host cell, each vector
should harbor a different antibiotic selection marker. A releasable C-
terminal clasp is usually introduced to the heterodimeric subunits of
integrin headpiece or extracellular domains to express the covalently
linked heterodimer in the same host. The release of this C-terminal
clasp by specific protease cleavage can be used to mimic the integrin
activation or for crystallization studies.
In the present study, we set up the constructs of integrin αIIbβ3
headpiece in which a releasable artificial clasp was flanked at the
C-terminal of truncated α- and β-subunits to mimic the integrin leg
stalk interaction to help generate the formal heterodimer during
overexpression [7]. The 30-residue ACID and BASE peptides each
with one cysteine mutation, which can form α-helical coiled-coils
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Lab Note
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with a pair of disulfide bond during the secretion expression, were
fused to the C-terminal of αIIb and β3, respectively, to help assemble
heterodimeric subunits into one entity. TEV cleavage sites were
designed and inserted between integrin subunits and coiled-coils. The
TEV sites were inserted before the ACID and BASE peptides and an
incorporated 6×-histidine tag was constructed at the C-terminal of
BASE peptide for removing the coiled-coils (Fig. 1A). The overlap
extension polymerase chain reaction (PCR) fragments of αIIb and
β3 subunits were inserted into the vector pcDNA 3.1/Hygro (−)
and pEF1-puro, respectively, for co-transfection. Then HEK 293T
cells were used to identify the constructs by transient transfection,
metabolic labeling and immunoprecipitation [8]. The 35 S-labeled
immunoprecipitation method was used to identify the disulfide bond
formation in coiled-coils, the intersubunit linkage quality, and the
secretion levels of clasped subunits into the medium. The results
indicated that αIIb and β3 subunits were expressed into one entity in
the same host like that under physiological status. When transfected
into HEK 293T cells, this soluble, clasped version of αIIbβ3 headpiece
was expressed as a secreted ∼120-kDa covalent heterodimer without
aggregates, shown on nonreducing gel, while on reducing gel it was
converted into a ∼75-kDa αIIb subunit and a ∼50-kDa β3 subunit
(Fig. 1B).
After identification of the construct strategy, the stable cell line of
CHO lec 3.2.8.1 was generated to overexpress the target protein by
long-term secretory overexpression [9]. Stable expression starts with
a transgene that has been stably integrated into the host cell’s genome.
The transgene expression level of the stably transfected cell line may
vary gradually. After several generations, no transgene expression
can be observed in that the coding sequence or the promoter of the
transgene may be destroyed. In this study, we found that the expand-
ing and screening of 24 clonal cell lines via ELISA after single cell
sorting is very efficient and a key step to get the highest expression
clones, which will affect the subsequent expression level. After two
generations via 24-well plate cultures, the same ELISA quantification
was performed again to test the expression level and finally to obtain
1
2 Long-term overexpression of human αIIbβ3 headpiece
Figure 1. Co-expression construct design and identification (A) Headpiece
portion of αIIb (1-621) and β3 (1-472) subunits were fused with 30-residue
ACID and BASE peptides, respectively, with one cysteine mutation. In both
subunits, the seven-amino acid recognition sequences for TEV protease,
flanked by short linkers, were inserted before the ACID and BASE peptides.
An incorporated 6×-histidine tag was constructed at the C-terminal of BASE
peptide. (B) Construct identification by metabolic labeling and immunopre-
cipitation. On nonreducing gel, α and β subunits assemble into one entity.
On reducing gel, the top bands are α subunits and the lower ones are β3
subunits and a clasped version of αIIbβ3 headpiece was expressed as a
secreted ∼120 kDa covalent heterodimer. They were converted into a ∼75-
kDa αIIb subunit and a ∼50-kDa β3 subunit on reducing gel. Other integrins
were used to identify the strategy.
the stable expression cell line. The purification strategies after stable
expression are flexible according to the transgene construct that
is transformed into the clonal cells. In the case of this protocol,
an incorporated 6×-histidine tag was constructed at the C-terminal
of one subunit, which allowed for the immobilized metal affinity
chromatography. Alternatively, target proteins purified by affinity
antibody columns are purer and of better yield as compared with
those of other methods. The 6×-histidine tag is used to remove the
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Figure 2. αIIbβ3 headpiece purification (A) AP2 antibody column purifica-
tion followed by FPLC Superdex 200. (B) Coiled-coil tag was removed by
TEV digestion and αIIbβ3 headpiece was further purified by Ni-NTA affinity
chromatography and gel filtration.
coiled-coils after TEV cleavage. After AP2 antibody column purifi-
cation, we got the same secreted ∼120-kDa covalent heterodimer as
that from transient expression by HEK 293T cells (Fig. 2A). TEV
protease treatment followed by Ni-NTA affinity column was used
to remove the coiled coils. αIIbβ3 headpiece samples were finally
purified by Superdex 200 chromatography to get the ∼75-kDa αIIb
subunit and ∼50-kDa β3 subunit, resulting in a final yield of ∼0.5 mg
of heterodimeric headpiece per liter of medium with high purity
(Fig. 2B).
In summary, an efficient protocol to produce soluble αIIbβ3
headpiece protein in mammalian cell line CHO lec 3.2.8.1 was
established. Based on the adherent culture and secretion expression,
protein samples can be obtained by collecting and refilling medium
repeatedly every 1 week for a long time. The protocol is useful for
those that require long-term overexpression of the heterodimeric
proteins via secretory and adherent cell cultures but is not suitable
for non-secretory expression.
Funding
This work was supported by the grant from the Heilongjiang Key
Laboratory for Laboratory Animals and Comparative Medicine (No.
518001).
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