ISSN 0582-9879 Acta Biochimica et Biophysica Sinica 2004, 36 (2): 141–146 CN 31-1300/Q

Short Communication

Refolding and Characterization of Recombinant Human GST-PD-1

Fusion Protein Expressed in Escherichia coli

Da-Wei LI, Jian-Feng YU, Yong-Jing CHEN, Hong-Bing MA, Zheng-Fei WANG,
Yi-Bei ZHU, and Xue-Guang ZHANG*
( Biotechnology Research Institute, Soochow University, Suzhou 215007, China )

Abstract Programmed death-1 (PD-1) is a costimulatory molecule of CD28 family expressed on

activated T, B and myeloid cells. The engagement of PD-1 with its two ligands, PD-L1 and PD-L2, inhibits
proliferation of T cell and production of a series of its cytokines. The blockade of PD-1 pathway is involved

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in antiviral and antitumoral immunity. In this study, human PD-1 cDNA encoding extracellular domain was
amplified and cloned into expression plasmid pGEX-5x-3. The fusion protein GST-PD-1 was effectively
expressed in E. coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was per-
formed to obtain bioactive soluble GST-PD-1. Fusion protein of above 95% purity was acquired by a conve-
nient two-step purification using GST affinity and size exclusion columns. Furthermore, a PD-L1-dependent
in vitro bioassay method was set up to characterize GST-PD-1 bioactivity. The results suggested that GST-
PD-1 could competently block the interaction between PD-L1 and PD-1 and increase the production of IL-
2 and IFN- of phytohemagglutinin-activated T cells.

Key words costimulatory molecule; GST-PD-1; refolding

Optimal activation of T cells for clonal expansion role in the binding of PD-1 to its two known ligands, PD-
depends on two distinct signals from antigen presenting L1 [7,8] and PD-L2 [9,10]. PD-1 is mainly expressed on
cells. One is the specific antigen delivered through the T activated T, B and myeloid lineage cells [11]. Ligation of
cell receptor by specific peptides in the context of major PD-1 with its two ligands inhibits the proliferation of T
histocompatibility proteins on antigen presenting cells, cells and its production of cytokines, especially IL-2 and
whereas the other is triggered through a distinct T cell IFN- [8,9]. Mice deficient in PD-1 suffered from lupus-
surface molecule. The B7-CD28 superfamily, one of the like glomerulonephritis and/or arthritis [12] in C57BL/6
best-characterized costimulatory families, not only background, or fatal autoimmune dilated cardiomyopathy
provides critical positive second signals to initiate and [13] in BALB/c background, which suggests negative
sustain T cell response, but also contributes key negative regulatory costimulator PD-1 is critical for the main-
second signals to down-regulation and termination of T tenance of peripheral tolerance and its deficiency could
cell response [1–3]. induce various autoimmune diseases [14]. Furthermore,
Programmed death-1 (PD-1), a recently described mem- blockade of PD-1 engagement accelerated a series of
ber of CD28 family, is a 55 kD type I transmembrane autoimmune diseases including autoimmune diabetes [15],
glycoprotein of Ig superfamily with a single extracellular experimental autoimmune encephalomyelitis (EAE) [16]
IgV-like domain [4–6]. This domain plays an important and graft-versus-host disease [17]. However some recent
studies also indicated that blockade of PD-1 pathway was
Received: September 8, 2003 Accepted: December 4, 2003
helpful to antiviral and antitumoral immunity [18,19]. A
This work was supported by a grant from the Major State Basic report proved that PD-L1 expression on tumor cells could
Research Development Program of China (No. 2001CB51003) and the serve as a potent mechanism for escaping from host
Key Medical Laboratory of Jiangsu Province
*Corresponding author: Tel, 86-512-65125011; Fax, 86-512-
immune responses and that effective blockade of the
65104908; E-mail, [email protected] interaction between PD-1 and PD-L1 was able to specifi-
142 Acta Biochimica et Biophysica Sinica Vol. 36, No. 2

cally kill the tumor cells by activated CTLs [18]. Another lished human PD-1 cDNA sequence, two oligonucleotide
study suggested that PD-1 played an important role in T primers (sense, 5'-TAAGGGATCCATTGGCGGCCAG-
cell tolerance at the effector phase [19]. Therefore, PD-1 GATGGTTC-3'; antisense, 5'-TAGGAATTCATTTG-
receptor may be of a promising target in control of virus GAACTGGCCGGCTGGCCTGGGTGA-3') were syn-
infection and immunotherapy of malignant cancers. thesized (Sangon Biotech., Shanghai). The first strand
In this report, we described a protocol for high-level cDNA was prepared by annealing 1–3 g total RNA, 0.5
expression and purification of the PD-1 extracellular g AMV reverse transcriptase (TaKaRa, Japan) and ran-
domain as GST fusion protein from Escherichia coli. dom primer (50mM) according to manufacturer’s
Furthermore, a PD-L1-dependent in vitro bioassay instructions. The mature extracellular domain sequence
method was set up to characterize GST-PD-1 bioactivity. of PD-1 was then amplified by PCR in 50 l reaction mix-
ture containing 1 g cDNA, 2.5 u LA Taq DNA polymerase
(TaKaRa), diluted buffer, nucleotides (2.5 mM/per) and
PD-1 specific primer (50 mM) sets. The reaction mixture
Materials and Methods
was denatured at 94 for 40 s, annealed at 58 for 45
s, and polymerized at 72 for 40 s. 32 cycles were per-
Materials
formed and followed by a 10 min extension at 72 . The

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Plasmid and bacteria Expression plasmid pGEX-5x-3 derived DNA fragments were digested with BamHI and
was originally purchased from Pharmacia (Sweden). EcoRI, and then cloned into similarly digested plasmid
Retrovirus vector pGEZ-Term and complementary pGEX-5x-3 under the control of tac promoter. The ex-
vectors, pHIT456 and pHIT60, were kindly provided by pression vector pGEX-5x-3-PD-1 was confirmed by ana-
Prof. Serfling E. (University of Wurzburg). Top10 and lysis of restriction digestion and DNA sequencing (BioAsia,
BL21 (DE3) used as hosts for cloning and gene expres- Shanghai), and then transformed into E. coli BL21 (DE3)
sion were from Invitrogen (USA) and Stratagene (USA). by CaCl2 method.
Enzymes and reagents All general laboratory chemicals
E. coli expression of GST-PD-1 fusion protein
(>99.9%) were obtained from Sigma or locally procured.
Restriction endonucleases and T4 ligase were purchased E. coli strain harboring the pGEX-5x-3-PD-1 was
from TaKaRa Biotech. (Japan). Yeast extract and peptone grown in 2 ml culture medium containing 1% glucose, 50
were from Oxford (USA). Trizol, RPMI 1640 and other mg/L ampicillin and 34 mg/L chloramphenicol for over-
cell culture reagents were from Gibco BRL (USA). night at 37 . The colony was expanded by inoculating
Protein purification materials and apparatus were from into 500 ml LB media at 37 till the A600 of culture reached
Pharmacia and Bio-Rad (USA). Polyclonal sheep anti- 1.0. The production of recombinant GST-PD-1 was in-
human PD-1 antibody and mouse anti-human PE- duced by 1 mM isopropylthiogalactosidase (IPTG). After
conjugated PD-L1 monoclonal antibody were purchased growing for 5 h, cells were harvested by centrifugation,
from Santa Cruz and eBioscience (USA). ELISA kits for then resuspended in 25 mM NaCl, 50 mM Tris-HCl, pH 8.
quantitative analysis of IL-2 and IFN- were purchased 0, 1 mM dithiothreitol (DTT) and 1 mM PMSF, finally
from Diaclone (France). broken by super-sonication for 30 min (100 W, 10-s pulse
with 10-s interval). Inclusion bodies were obtained by cen-
Cell culture
trifugation at 10,000 g for 15 min, and washed several
L929 cells were propagated in RPMI 1640 medium sup- times with 2.5 M urea and 1% Triton X-100.
plemented with 25 mM HEPES, 10% fatal bovine serum,
Refolding and purification procedures for recombinant
2 mM glutamine, 100 mg/L penicillin and 100 mg/L
GST-PD-1
streptomycine. 500 mg/L Zeocin (Invitrogen) was added
to the growth medium of PD-L1 transfected L929 cells. Cell pellet was resuspended in 50 ml solution with 8 M
urea, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM
Cloning of PD-1 cDNA and construction of expression
EDTA and DTT, and 1 mM PMSF, and the suspension
vector
was stirred slowly for overnight at 4 to reduce and
Healthy human peripheral blood T cells were activated denature the expressed GST-PD-1 fusion protein. The so-
in vitro for 2 d with 10 mg/L phytohemagglutinin (PHA, lution was centrifuged at 10,000 g for 30 min and the
Sigma), then total RNA was prepared using Trizol accor- insoluble pellet was discarded. The supernatant was slowly
ding to manufacturer’s instructions. Based on the pub- dropped into 450 ml refolding solution with 1 mM reduced
Feb., 2004 Da-Wei LI et al.: Refolding and Characterization of Recombinant Human GST-PD-1 Fusion Protein 143

glutathione, 0.2 mM oxidized glutathione, 2 M urea in 50 vectors pHIT456 and pHIT60, was cotransfected pack-
mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% Tween 20 and 1 age cell 293T using LipofectAmine kit (Invitrogen). Then
mM PMSF, then vigorously stirred. The refolding mix- the supernatant of 293T culture was used to infect L929
ture was incubated for 3 d at 4 , centrifuged to remove cells. Stable transformants were selected in medium con-
insoluble materials, and then dialyzed against 5 L solution taining 500 mg/L Zeocin for 2 weeks till separated resis-
A (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM tant cell lines grew up. After another week’s culture sev-
KH2PO4, pH 7.3) for 18 h at 4 using 10 kD molecular eral cell lines expressing PD-L1 on cell surface were
weight cutoff dialysis tubing. After centrifugation to re- screened by flow cytometry using a PE-conjugated
move insoluble materials, 250 ml supernatant was filtered anti-human PD-L1 monoclonal antibody following the
and loaded onto a 1 ml GSTrap FF affinity chromato- manufacturer’s directions. A nonspecific mouse mono-
graphy column (0.7 cm×2.5 cm, Pharmacia) previously clonal antibody of the same type was used as negative
equilibrated with solution A, at a flow rate of 1 ml/min. control. Labeled cells were analyzed for positive fluores-
Bound proteins were eluted with 50 mM Tris-HCl, pH cence using a Coulter flow cytometer. A mock transfect-
8.0, and 10 mM reduced glutathione. Eluted fractions were ed L929 cell line was prepared using the similar procedures.
collected and further loaded onto a Bio-Gel P-100 column IL-2 and IFN- production bioassays
(1.5 cm×70 cm, Bio-Rad) equilibrated in 50 mM Tris-

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HCl, pH 8.0, and 250 mM NaCl, at a flow rate of 0.06 Peripheral blood mononuclear cells of healthy donors
ml/min. The pooled fractions containing recombinant GST- were isolated by Ficoll-Hypaque gradient centrifugation
PD-1 protein were filtered through sterile 0.22 m mem- passed through a nylon wool column to obtain purified
brane and stored at –70 . Protein concentration was T cells (about 85% CD3+ T cells). T responder cells and
determined by measuring absorbance at 280 nm and Lowy PD-L1 or mock transfected L929 stimulator cells pretreated
protein assay using bovine serum albumin as standard. with 50 g/10 7 cells MMC were added to each well of
a 96-well plate at concentrations of 4×105 cells/L and
SDS-PAGE and Western blot 2×105 cells/L, respectively (a final volume of 250 l per
Protein samples were analyzed by discontinuous SDS- well). PHA was added at a concentration of 10 mg/L.
PAGE according to Laemmli (1970). Sample was de- 1 mg/L GST-PD-1 or GST control protein were separate-
natured in sample buffer by boiling for 10 min, and then ly added to PD-L1 transfected cell. Samples were ana-
used for electrophoresis on a 12% SDS/polyacrylamide lyzed in triplicate. The plates were incubated for 48 h
gel. Protein staining was performed with Coomassie bril- at 37 in a humidified tissue culture incubator. 100 l
liant blue. For Western blot, the proteins were transferred aliquot from each well was analyzed for secreted IL-2 and
from the unstained SDS/polyacrylamide gel to nitro- IFN- using corresponding kits following the manu-
cellulose membrane by electrophoresis (0.65 mA/cm2) for facturer’s directions. Optical densities of the plates were
2 h. The filter was blocked overnight at 4 using block- read at 450 nm using a microplate reader and were con-
ing solution. After washed 3 times, it was incubated with verted to doses of IL-2 and IFN- in ng/L according to
sheep anti-human PD-1 polyclonal antibody for 1 h at room parallel standard curves.
temperature and then washed again. HRP-conjugated rab-
bit secondary antibody was added. 1 h later, the filter was
repeatedly washed, and the specific proteins were detec- Results
ted by color reaction with the appropriate substrate.
Construction of vector for expression of the
Construction of PD-L1 transfected L929 cell lines GST-PD-1 fusion protein
Full-length PD-L1 cDNA was cloned from human heart The extracellular domain coding sequence of PD-1 was
cDNA library (Invitrogen) by PCR using the following two cloned using RT-PCR from PHA activated peripheral blood
p r i m e r s ( s e n s e , 5 ' - TA C T G C A G A A G AT G A G - T cells. This fragment was fused to the downstream of
GATATTTGCTGTC-3'; antisense, 5'-ATTGAATTCT- GST sequence on pGEX-5x-3 to yield expression vector
TACGTCTCCTCCAAATGTG-3'). The 0.9 kb PD-L1 pGEX-5x-3-PD-1. The enzyme digestion and sequence
cDNA fragment was digested with PstI and EcoRI and analysis results showed that the cloned gene fragment
cloned into similarly cut retrovirus vector pGEZ-Term. consists with the published data (GenBank accession No.
The expression vector containing the PD-L1 cDNA U64863). The construction was used to express a 149
sequence, together with the other two complementary amino acid (19–167 aa) PD-1 extracellular domain fused
144 Acta Biochimica et Biophysica Sinica Vol. 36, No. 2

to GST protein. centrifugation, and almost all of the GST-PD-1 was in the
insoluble fraction. After denaturing and refolding of in-
Expression and purification of GST-PD-1 fusion
clusion bodies, the supernatant was fractioned on a GST
protein
affinity column. A single peak was eluted by reduced glu-
After the expression plasmid pGEX-5x-3-PD-1 was tathione solution (Fig. 2). For further purification of re-
transformed into E. coli BL21 (DE3), GST-PD-1 fusion folded proteins, an exclusion chromatography was used
protein was highly expressed after induced by IPTG and and the peak eluted at approximately 10 h was collected
the optimum expression in shake flasks occurred after (Fig. 3). SDS-PAGE analysis showed the fraction con-
5–7 h induction with 1 mM IPTG. The reducing SDS- sisted primarily of a 45 kD protein [Fig. 1(A)]. Then the
PAGE analysis showed that molecular weight of GST- expressed GST-PD-1 protein was confirmed by Western
PD-1 was about 45 kD which agrees with the predicted blot [Fig. 1(B)]. This convenient two-step procedure
value from the gene sequence (Fig. 1). After expression resulted in about 11.5 mg fusion protein from 500 ml E.
induction, soluble and insoluble proteins of the cell pellet coli culture with a purity higher than 95% according to
were separated from each other after sonication and densitometer scanning of SDS-PAGE gels. A summary of
fusion protein purification procedure is given in Table 1.

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In vitro bioassays of the GST-PD-1 fusion protein
The bioactivity of GST-PD-1 was determined by using
a cell-based bioassay with human peripheral T cells as

Fig. 1 SDS-PAGE and Western blot of recombinant

GST-PD-1 fusion protein
(A) SDS-PAGE. 1, whole cell lysates (un-induced); 2–5, whole cell lysates Fig. 2 GST affinity chromatography of refolded GST-PD-1
(induced for 1, 3, 5 and 7 h); 6 and 7, purified GST-PD-1 fusion protein after GST The sterile filtered sample was applied to a 1 ml GSTrap FF equilibrated in 140
affinity and gel chromatography; 8, whole cytosolic proteins containing GST mM NaCl, 12.7 mM KCl, 10 mM Na2HPO4, 1.8 mmol /L KH2PO4, pH 7.3, with
expression proteins; 9, purified GST control protein. All of above samples were a flow rate of 1 ml/min. The column was washed with the same buffer until the
directly subjected to 12% SDS-PAGE with the size control of molecular weight absorbance at 280 nm returned to base line. Elution was performed with 50 mM
marker (Lane M). (B) Western blot analysis of GST-PD-1. 1, whole cell lysates Tris-HCl, pH 8.0, and 10 mM reduced glutathione. Purified GST-PD-1 protein
(un-induced); 2, purified GST-PD-1 fusion protein. was collected from the elution peak.

Table 1 Purification of rhGST-PD-1

Purification step Total protein (mg) rhGST- PD- 1 purity (%) rhGST- PD- 1 (mg) Recovery (%)

Bacteria total protein 228.2 ± 15.6 20 45.6 ± 3.1 100

Soluble inclusion body 48.7 ± 3.3 45 21.9 ± 1.5 48

GSTrap FF 15.2 ± 1.0 90 13.7± 0.9 30

Bio- Gel P- 100 11.5 ± 0.7 95 10.9± 0.7 24

Feb., 2004 Da-Wei LI et al.: Refolding and Characterization of Recombinant Human GST-PD-1 Fusion Protein 145

Discussion

This study demonstrated the feasibility of producing

biologically active GST-PD-1 fusion protein in bacteria.
The GST-PD-1 was generated using the well-known
glutathione S-transferase fusion system, which provided
high-level expression and following easy purification [20].
But our experiments using pGEX-5x-3 expression vector
showed that almost all of the GST-PD-1 protein was in
inclusion bodies. Unfortunately, all attempts to induce
Fig. 3 Purification of GST-PD-1 by size exclusion column soluble active product by altering expression conditions
The pooled fractions from affinity chromatography were loaded onto a Bio-Gel (temperature, induction time, IPTG concentration, cell-
P-100 column equilibrated in 50 mM Tris-HCl, pH 8.0 and 250 mM NaCl. The density aeration conditions, or pH of culture) were proved
further details were described in the text. The major absorbance peak of pooled
unsuccessful. Therefore, we had to denature and refold
fractions contained active GST-PD-1 fusion protein.
the inclusion bodies to obtain soluble biologically active

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protein. In the process of refolding, a series of pro-
responder cells and mock or PD-L1 transfected L929 cell cedures were tested and an optimal refolding condition
lines as stimulator cells (Fig. 4). It was found that T cells (see methods) for producing a high-quality, good-yield of
secreted IL-2 and IFN- when stimulated by mock trans- GST-PD-1 was determined. To elevate the purity of GST-
fected L929 cells in presence of PHA, similar to the PD-1, an exclusion column was used to separate GST-
response of freshly isolated T cells (data not shown). PD-1 from contaminations derived from GST affinity
However PD-L1 transfected L929 cells significantly chromatography. The result indicated that the two-step
inhibited the production of these cytokines of PHA purification is effective with GST-PD-1 purity higher than
activated T cells at different PHA concentrations. The 95%.
optimum concentration of PHA was determined to be Increasing evidences suggested that the engagement of
10 mg/L. When GST-PD-1 fusion protein was added to PD-L1 or PD-L2 with PD-1 could inhibit production of a
the cultures of PD-L1 transfected L929 cells, the series of cytokines, especially IL-2 and IFN- , of pre-
inhibitory effects were gradually reversed with the in- activated peripheral blood T cells [8,9]. PD-1 receptor could
creasing of GST-PD-1 concentration (data not shown). be induced and up-regulated on PHA activated T cells and
GST-PD-1 reversed the inhibitory effects at 1 mg/L in the peaked at 48 h [21]. To evaluate bioactivity of GST-PD-1,
assay. Purified GST protein was used as negative control. we constructed a PD-L1 transfected L929 cell line.
Compared with mock transfected L929 cells, PD-L1 trans-
fected cells could inhibit the production of IL-2 and
IFN- of PHA-activated T cells. The result showed cell-
surface-associated PD-L1 could interact with PD-1 up-
regulated expressions on T cells (data not shown) and
inhibit production of IL-2 and IFN- , which was con-
sistent with previous report [22]. Interestingly, the higher
level IFN- was produced because PHA-activated T cells
were predominantly CD8+ phenotype with their intrinsic
inability to produce significant levels of IL-2 [23]. More
importantly, GST-PD-1 could competently block the
interaction of PD-L1 with its receptor (PD-1), and increase
Fig. 4 Bioassays of GST-PD-1 fusion protein T cell cytokine synthesis. Therefore, bacterial derived
(A) IL-2. (B) IFN- . 1, T cells (1×105 per well) and PD-L1 and mock transfected GST-PD-1 was a potent inhibitor of cell-surface-asso-
L929 cells (5×104 per well) were incubated in the presence of 10 mg/L PHA; ciated PD-L1 in the T cell IL-2 and IFN- production
2, GST-PD-1 (1 mg/L) and GST control proteins (1 mg/L) were added to the
bioassays.
cultures of PD-L1 transfections. After 24 h, supernatants were assayed for secreted
IL-2 and IFN- by ELISA. Data are means of triplicate wells from a representative
The extracellular domain of PD-1 contains four N-linked
experiment. *P<0.05 vs. Mock-L929+T (control); #P<0.05 vs. GST (control). glycosylation sites, and the protein is believed to be gly-
146 Acta Biochimica et Biophysica Sinica Vol. 36, No. 2

cosylated in vivo [6]. The fact that bacterially derived GST- Iwai Y et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation.
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