Protein Expression and Purification 99 (2014) 50–57
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Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells Aleksandra Sokolowska-Wedzina a,1, Aleksandra Borek a,1, Julia Chudzian a, Piotr Jakimowicz b, Malgorzata Zakrzewska a, Jacek Otlewski a,⇑ a b
Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland Faculty of Biotechnology, Department of Protein Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 8 January 2014 and in revised form 26 March 2014 Available online 13 April 2014 Keywords: FGFR CHO Protein expression Glycosylation
a b s t r a c t The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2bFc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Fibroblast growth factor (FGF)2 signaling regulates numerous important biological processes including embryonic development, tissue homeostasis, angiogenesis and general metabolism. The biological effects of FGFs observed in target cells are mediated through specific cell-surface tyrosine kinase receptors, called fibroblast growth factor receptors (FGFRs) [1,2]. Four FGF receptors have been identified to date, FGFR-1 through FGFR-4, which share between 55% and 72% homology at the amino acid sequence level [3]. The prototypical FGFR consists of an extracellular ligand-binding region composed of three Ig-like domains (D1, D2, D3), a unique acidic serine-rich region (acid box), a transmembrane domain, and a well conserved split tyrosine kinase domain responsible for the activation of downstream signaling and biological responses [4,5]. ⇑ Corresponding author. Tel.: +48 71 375 2824. E-mail address: [email protected] (J. Otlewski). Equally contributing authors. 2 Abbreviations used: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; ECD, extracellular domain; SSP, secretion signal peptide; NSP, native signal peptide; PCR, polymerase chain reaction; PEI, polyethyleneimine; PBS, phosphate buffered saline. 1
http://dx.doi.org/10.1016/j.pep.2014.03.012 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
The first Ig-like domain (D1) and the acid box are involved in the auto-inhibitory function of the receptors, whereas the second and third Ig domains are critical for the ligand binding and its specificity. Regulation of the ligand-binding specificity of FGFR is essential for the control of FGF signaling and is primarily achieved by alternative splicing of FGFR1-3 transcripts [6]. An alternative splicing event in FGFR1-3 involving the exon encoding the C-terminal region of D3 results in two receptor isoforms (IIIb and IIIc) of different ligand-binding specificities. Additional alternative splicing events result in FGFR isoforms that lack D1 (in FGFR1 and 2), D1 and the D1–D2 linker (in FGFR2), or the D1–D2 linker (in FGFR3) [4]. Overexpression of FGFRs has been reported in a wide variety of tumors, such as breast, bladder, prostate, endometrial, lung and hematologic cancers, and has been associated with tumor progression and poor patient prognosis [7–9]. Depending on the localization and cancer type, different FGFRs are overexpressed. For example, elevated levels of FGFR1 [8,10–12], FGFR2 [13], and FGFR4 [14] are found in breast cancers, of FGFR2 in gastric cancers [15], overexpression of FGFR1 and FGFR3 is associated with papillary thyroid carcinoma [16,17], and of FGFR1 and FGFR4 with prostate cancers [18]. Moreover, low levels of FGFR are found on noncancer cells in the tumor vicinity. Therefore, FGFRs represent
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A. Sokolowska-Wedzina et al. / Protein Expression and Purification 99 (2014) 50–57 Table 1 Constructed plasmids encoding extracellular domain of FGFRs fused with Fc fragment. Plasmid name
Plasmid size
Amino acids of FGFRs
D3 domain splicing variant
Gene ID
RefSeq number (mRNA)
RefSeq number (protein)
pLEV113-ECD_FGFR1a-Fc pLEV113-ECD_FGFR2a-Fc pLEV113-ECD_FGFR3a-Fc pLEV113-ECD_FGFR4a-Fc pLEV113-ECD_FGFR1b-Fc pLEV113-ECD_FGFR2b-Fc pLEV113-ECD_FGFR3b-Fc pLEV113-ECD_FGFR4b-Fc
6924 bp 6936 bp 6927 bp 6912 bp 6656 bp 6669 bp 6618 bp 6621 bp
22–374 22–377 23–375 22–369 22–24, 120–374 22–36, 126–377 23, 124–375 119–369
IIIc IIIc IIIc – IIIc IIIc IIIc –
2260 2263 2261 2264 2260 2263 2261 2264
NM_015850.3 NM_000141.4 NM_000142.4 NM_002011.3 NM_015850.3 NM_000141.4 NM_000142.4 NM_002011.3
NP_056934.2 NP_000132.3 NP_000133.1 NP_002002.3 NP_056934.2 NP_000132.3 NP_000133.1 NP_002002.3
‘‘a’’ in the name of construct indicates whole extracellular domain of FGFR (containing D1, acid box, D2, D3), ‘‘b’’ indicates shortened fragment of extracellular domain, lacking D1 (acid box, D2, D3). ECD – extracellular domain.
an attractive target for anti-tumor therapy. Until now, small-molecule tyrosine kinase inhibitors and several antibodies have been tested to target FGFRs in various types of cancer [8,9,19]. In this study, we have cloned, expressed and purified variants of Fc-tagged extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2bFc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGF R4b-Fc, Table 1). Such fusion proteins are convenient molecular targets for the selection of a range of FGFR-specific antibodies. Materials and methods Construction of ECD_FGFR-Fc expression plasmids An expression construct ECD_FGFR1a-Fc was prepared in the mammalian expression vector pLEV113 (pLEV113-ECD_FGFR1aFc) by LakePharma (Belmont, CA). It contained a DNA fragment corresponding to the extracellular domain (ECD) of FGFR1 isoform IIIc (residues 22–374), which was preceded by a secretion signal peptide and followed by the sequence of human IgG1 Fc domain, with a nine-amino acid glycine-rich linker between the ECD-FGFR1 and Fc. cDNAs encoding full-length sequences of FGFR2, FGFR3 and FGFR4 were kindly provided by Antoni Wiedlocha’s Laboratory (Oslo University Hospital). These cDNAs together with pLEV 113-ECD_FGFR1a-Fc were then used to prepare expression constructs: pLEV113-ECD_FGFR1b-Fc, pLEV113-ECD_FGFR2a-Fc, pLEV 113-ECD_FGFR2b-Fc, pLEV113-ECD_FGFR3a-Fc, pLEV1113-ECD_ FGFR3b-Fc, pLEV113-ECD_FGFR4a-Fc, pLEV113-ECD_FGFR4b-Fc
(Table 1). All PCR primers used for plasmid preparation are summarized in Tables 2 and 3. The plasmids were verified by sequencing, amplified and purified using Plasmid Maxi Kit (Qiagen). Cell culture and transfection CHO-S cells (Invitrogen), adapted for growth in suspension, were cultured in serum- and protein-free PowerCHO-2CD medium (Lonza) supplemented with 8 mM L-glutamine and 1 penicillin/streptomycin solution. Cells were grown at 37 °C in a shaking incubator (140 rpm) with 8% CO2. Routine subculturing was conducted every 2–3 days at seeding density of 0.1– 0.3 106 cells/ml. One day prior to transfection, cells were seeded at a density of 0.6 106 cells/ml in culture medium. On the day of transfection CHO-S cells were centrifuged and cell pellet was resuspended in serum- and protein-free ProCHO4 medium at 2 106 cells/ml. Plasmid (1 mg/ml) and PEI (1 mg/ml) stocks were diluted separately in 150 mM NaCl, mixed at a 1:4 (w:w) ratio, incubated at RT for 10 min, and added to the cell culture (1.25 lg DNA per 1 106 cells, total volume added was 1/10 of the volume of transfected culture). Cells were incubated for 4 h in standard conditions (37 °C, 140 rpm, 8% CO2). After this time the culture was diluted with an equal volume of PowerCHO-2CD supplemented with 8 mM L-glutamine and 2 penicillin/streptomycin solution to obtain the cell density of about 1 106 cells/ml, and incubated at 31 °C. At day 2 following transfection the culture was supplemented with 4 mM L-glutamine and finally harvested at day 6.
Table 2 Primers used for preparation of pLEV113-ECD_FGFRa-Fc plasmids. Symbol
Name
A
FGFR1a-Kpn2I-Fc-F
5 CCTGTACCTGGAGTCCGGAGGAGGTGGTGCAGG 3
FGFR1a-Kpn2I-Fc-R
50 CCTGCACCACCTCCTCCGGACTCCAGGTACAGG 30
MutFGFR2-F
50 GTGAATGTCACAGATGCCATCTCATCAGGTGATGATGAGGAT GACACCGATGG 30
MutFGFR2-R
50 CCATCGGTGTCATCCTCATCATCACCTGATGAGATGGCATCT GTGACATTCAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCGGCCCT CCTTCAGTTTAGTTGAGG 30
B
C
FGFR2a-SSP-F
D
FGFR2a-Kpn2I-R
E
FGFR3a-SSP-F
F
FGFR3a-Kpn2I-R
G
Sequence 0
Specification 0
Introduction of Kpn2I restriction site Kpn2I restriction site removal
Addition of SSP fragment
50 tacgTCCGGACTCCAGGTAGTCTGGGGAAGC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGAGTCCTT GGGGACGGAGCAGC 30
Addition of Kpn2I restriction site
50 tacgTCCGGAGCCTGCATACACACTGCCCGCC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCTGGAGGC CTCTGAGGAAGTGG 30
Addition of Kpn2I restriction site
FGFR4a-SSP-F
H
FGFR4a-Kpn2I-R
50 tacgTCCGGAGTCCGTATACCTGGCCTCGGG 30
Addition of Kpn2I restriction site
I
HindIII-SSP-F
50 ctccAAGCTTTGAACCACCATGGAATGGAGCTGGGTCTTTCTCTT CTTCCTGTCAGTAACGACTGG 30
Addition of C-terminal fragment of SSP with HindIII restriction site
SSP – secretion signal peptide.
Addition of SSP fragment
Addition of SSP fragment
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Table 3 Primers used for preparation of pLEV113-ECD_FGFRb-Fc constructs. Symbol J K L M N O P R S T
Name FGFR1b-F FGFR1bSSP-F FGFR2b-1-F FGFR2b-2-F FGFR2bSSP-F FGFR3b-F FGFR3bSSP-F FGFR4b-F FGFR4bSSP-F Fc-Not1-R
Sequence 0
Specification 0
5 GATGCTCTCCCCTCCTCGGAGGATGATG 3 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCAGGCCGTCCGATGCTCTCCCCTCCTCGGAGGATGATG 30 50 GATGCCATCTCATCAGGTGATGATGAGGATGACAC 30 50 CTTCAGTTTAGTTGAGGATACCACATTAGAGCCAGAAGATGCCATCTCATCAGGTGATGATGAGGATGACAC 30 50 TTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCGGCCCTCCTTCAGTTTAGTTGAGGATACCACATTAGAGCC 30 50 GACGCTCCATCCTCGGGAGATGACGAAGAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGAGGACGCTCCATCCTCGGGAGATGACGAAGAC 30
Cloning D2D3-Fc fragment Addition of SSP fragment
50 GACTCCTTGACCTCCAGCAACGATGATGAGGAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGACTCCTTGACCTCCAGCAACGATGATGAGGAC 30
Cloning D2D3-Fc fragment Addition of SSP fragment
50 tcgaGCGGCCGCTCATTTACCCGGAGAC 30
Cloning of ECD-FGFR-Fc
Cloning D2D3-Fc fragment Addition of first 15 amino acids of FGFR2 Addition of SSP fragment Cloning D2D3-Fc fragment Addition of SSP fragment
SSP – secretion signal peptide.
Fig. 1. Construction of pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc plasmids. SSP – secretion signal peptide, L – linker, NSP – native signal peptide.
Purification of FGFRs-Fc Cell culture was harvested by centrifugation at 15,000 g for 20 min. The resultant supernatant was filtered through 0.22micron filter units and loaded onto a Protein A Sepharose column (GE Healthcare) pre-equilibrated with PBS. The unbound fraction was removed and the resin was washed first with 500 ml of washing buffer A: 30 mM Na2HPO4, 20 mM NaH2PO4, 350 mM NaCl, 0.1% Tween 20, 2 mM EDTA, pH 7.4 and then with 500 ml of washing buffer B: 30 mM Na2HPO4, 20 mM NaH2PO4, 650 mM NaCl, 2 mM EDTA, pH 7.4. Proteins specifically bound to the resin were
eluted with 100 mM triethylamine pH 11.5, and then immediately neutralized with 1 M Tris–HCl pH 7.2. Fractions with the highest protein concentrations were collected, dialyzed against PBS, and concentrated using Centriprep 10 K centrifugal filter units (Millipore). Protein purity was checked by SDS–PAGE and their identity was confirmed by mass spectrometry. Deglycosylation of ECD_FGFRs-Fc The recombinant proteins were treated with PNGase F according to the manufacturer’s instructions (New England BioLabs). In
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brief, 2 ll of 10 glycoprotein denaturing buffer was added to 2.5 lg of protein in 20 ll buffer and incubated at 95 °C for 10 min, then 3 ll of 10xG7 reaction buffer, 3 ll of NP-40 and 1 ll (500 U/ll) of PNGase F were added to each reaction mix and samples were incubated at 37 °C for 1 h. The effect of the deglycosylation on the molecular mass of the proteins was determined by SDS–PAGE and mass spectrometry.
co-crystallization. Whole-protein spectra were recorded in the high mass range with a 4800 Plus MALDI TOF/TOF (AB SCIEX) mass spectrometer. Next, N-terminal sequencing was done using MALDI-PDS-MS analysis for peptide mass range of 600–4000 Da.
SDS–PAGE and Western blotting
Construction of ECD_FGFR-Fc expression plasmids
The proteins were separated on SDS–PAGE 4–20% gradient gel (Pierce, Thermo Scientific) and either stained with Coomassie blue or electrotransferred to a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-FGFRs antibodies (Flg (S-16), Santa Cruz, sc-31169; Bek (H-80), Santa Cruz, sc-20735; FGFR3 (ZZ10), Santa Cruz, sc-73994; FGFR4 (A10), Santa Cruz, sc136988) and anti-Fc-HRP-conjugated antibody (KPL, 4-10-2020) in 1% BSA for 1.5 h at RT. After incubation with the primary antibodies the membrane was washed with 0.1% Tween 20 in PBS and incubated with the appropriate HRP-conjugated secondary antibodies in 1% BSA for 1 h at RT and finally washed with 0.1% Tween 20 in PBS. The blot was developed with the ECL reagent (Pierce, Thermo Scientific) according to the manufacturer’s instructions.
For expression of recombinant ECD_FGFR-Fc fusion proteins, plasmids pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc were constructed. The cloning procedure is presented in Fig. 1. Plasmid pLEV113-ECD_FGFR1a-Fc was modified by introduction of a Kpn2I restriction site (TCCGGA) in the linker region between the extracellular domain of FGFR1a and Fc using the pair of primers marked A (Table 2). The resulting plasmid was digested with HindIII/Kpn2I and named pLEV113-Kpn2I-Fc (Fig. 1A). Simultaneously, cDNAs encoding FGFR2 residues 22– 377, FGFR3 residues 23–375 or FGFR4 residues 22–369 were amplified by two rounds of PCR. The first PCR introduced at the 50 end a fragment encoding part of the extracellular targeting signal peptide (primers C, E, G, Table 2) and a Kpn2I site at the 30 end (primers D, F, H, Table 2). The second PCR completed the secretion signal peptide and introduced a HindIII site (primer I and D, F, H) (Fig. 1B). Additionally, FGFR2 cDNA was mutated to remove its own Kpn2I site (TCCGGA into TCAGGT) without changing amino acid sequence (pair of primers B, Table 2). Finally, the PCR products were digested and ligated into the HindIII/Kpn2I sites of the modified pLEV113-Kpn2I-Fc expression vector (Fig. 1C and 2). The
Mass spectrometry Protein samples were diluted with MALDI matrix (sinapinic acid 10 mg/ml in 50% acetonitrile, 0.1% TFA) at a ratio 1:2, 1:4 or 1:8 and deposited onto an Opti-TOF™ 384 Well Plate (AB SCIEX) for
Results and discussion
Fig. 2. Map of vector pLEV113 with ECD_FGFRs-Fc cloning site indicated. SSP – secretion signal peptide.
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Fig. 3. Cloning procedure for construction of pLEV113-ECD_FGFR1b-Fc, pLEV113-ECD_FGFR2b-Fc, pLEV113-ECD_FGFR3b-Fc, and pLEV113-ECD_FGFR4b-Fc plasmids. Sequence of FGFR2 D1 to be removed was determined according to naturally occurring variant of FGFR2 lacking D1 (RefSeq Number: NP_001138391.1, Isoform IIIb). SSP – secretion signal peptide, L – Linker.
sequences of recombinant plasmids pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc were confirmed by sequencing (LGC Genomics). For expression of respective proteins lacking the D1 domain, plasmids pLEV113-FGFR1b-Fc, pLEV113-FGFR2b-Fc, pLEV113FGFR3b-Fc, and pLEV113-FGFR4b-Fc were constructed. Their cloning procedure is presented in Fig. 3. DNA fragments corresponding to FGFR1 residues 22–24 and 120–374; FGFR2 residues 22–36 and 126–377; FGFR3 residues 23 and 124–375, and FGFR4 residues 119–369 were amplified with the downstream Fc sequence from
pLEV113-ECD_FGFRa-Fc plasmids by three rounds of PCR with one additional round for the ECD_FGFR2b-Fc construct. DNA sequences corresponding to the above fragments of all four constructs were amplified by the first PCR (primers J, L, O, R and T, Table 3). The second PCR introduced a fragment encoding the Nterminal part of the extracellular targeting signal peptide (primers K, P, S, and T, Table 3) of FGFR1b-Fc, FGFR3b-Fc, FGFR4b-Fc, and the first 15 amino acids of FGFR2b-Fc (primers M and T, Table 3). The additional PCR introduced a fragment encoding the N-terminal part of extracellular targeting signal peptide of FGFR2b-Fc (primers
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Fig. 4. Expression of recombinant ECD_FGFR-Fc proteins. As an example, expression of ECD_FGFR1a-Fc is presented. Culture supernatant was analyzed at each day of expression by (A) SDS–PAGE under reducing conditions followed by Coomassie blue staining and (B) Western blotting with anti-FGFR1 antibodies. (C) Cell growth and recombinant protein production by CHO-S cells. Histograms represent recombinant protein production expressed as band areas on anti-FGFR1 Western blots. The line represents cell growth expressed as viable cells/ml of culture. Additional glutamine supplementation on day 2 of protein expression is marked. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Purification of recombinant ECD_FGFR-Fc proteins. Purification steps were analyzed by (A) SDS–PAGE under reducing condition followed by Coomassie blue staining and (B) Western blotting with anti-FGFR4 antibodies. (C) Recombinant ECD-FGFR-Fc protein is isolated as a dimer. Purified ECD_FGFR4a-Fc was analyzed under non-reducing (sample buffer without b-mercaptoethanol and no heating) or reducing (sample buffer supplemented with b-mercaptoethanol, sample heated at 99 °C for 10 min) conditions, followed by Coomassie blue staining. Analysis of all purified recombinant ECD_FGFR-Fc proteins by (D) SDS–PAGE under reducing conditions followed by Coomassie blue staining, (E) Western blotting with anti-Fc antibodies, (F) Western blotting with specific anti-FGFR antibodies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
N and T, Table 3). The last PCR completed the secretion signal peptide and introduced a HindIII site to all constructs (primers I and T, Table 3) (Fig. 3A). Agarose gel extraction was performed after each round of PCR. Final PCR products were ligated into the HindIII/NotI sites of the pLEV113 vector (Fig. 3B and C). The sequences of pLEV113-FGFR1b-Fc, pLEV113-FGFR2b-Fc, pLEV113-FGFR3b-Fc, and pLEV113-FGFR4b-Fc plasmids were confirmed as above. Expression and purification of recombinant FGF receptors A mammalian cell transient transfection system was used to express fully glycosylated ECD_FGFR-Fc fusion proteins. The use of serum-free medium reduced the level of extraneous proteins in the culture medium and facilitated the detection and purification of secreted recombinant proteins [20–22]. The level of the recombinant proteins in culture medium during expression was monitored and analyzed by SDS gel electrophoresis in reducing conditions followed by Coomassie blue staining (Fig. 4A) and by
Table 4 Yields of individual ECD_FGFR-Fc protein preparations. Recombinant protein
Average protein yield [mg protein/l culture] ± SD (n = 3)
ECD_FGFR1a-Fc ECD_FGFR2a-Fc ECD_FGFR3a-Fc ECD_FGFR4a-Fc ECD_FGFR1b-Fc ECD_FGFR2b-Fc ECD_FGFR3b-Fc ECD_FGFR4b-Fc
20.0 ± 3.6 21.1 ± 1.8 18.3 ± 5.2 21.2 ± 2.7 12.7 ± 2.7 14.1 ± 4.1 14.8 ± 2.9 13.2 ± 4.3
Western blot analysis using specific anti-FGFR antibodies (Fig. 4B). Fig. 4C summarizes the time-course of cell growth and recombinant protein production in CHO-S cells for 6 days following transfection.
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Fig. 6. SDS–PAGE of native and deglycosylated recombinant ECD_FGFR-Fc proteins. Purified ECD_FGFR-Fc proteins were subjected to electrophoresis under denaturating conditions without or with prior PNGase F treatment.
Table 5 Characteristics of ECD_FGFR-Fc proteins. MS MW of proteins [kDa] (monomer)
SDS–PAGE MW of proteins [kDa] (monomer)
67.4
80.0
100–110
67.9
78.2
100–110
WSWVFLFFLSVTTGVHSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPP
66.5
78.5
100–110
ECD_FGFR4a-Fc
WSWVFLFFLSVTTGVHSLEASEEVELEPCLAPSLEQQEQELTVALGQPVRLCCGRA
66.9
79.3
100–110
ECD_FGFR1b-Fc
EWSWVFLFFLSVTTGVHSRPSDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSP
57.1
69.7
80–90
Recombinant protein
N-terminal sequence determined using MALDI-PSD-MS analysis
Calculated MW [kDa] (monomer)
ECD_FGFR1a-Fc
SWVFLFFLSVTTGVHSRPSPTLPEQAQPWGAPVEVESFLVHPGDLLQL
ECD_FGFR2a-Fc
EWSWVFLFFLSVTTGVHSRPSFSLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEVR
ECD_FGFR3a-Fc
ECD_FGFR2b-Fc
SWVFLFFLSVTTGVHSRPSFSLVEDTTLEPEDAISSGDDEDDTDGAEDFVSENSNN
58.1
68.4
80–90
ECD_FGFR3b-Fc
SWVFLFFLSVTTGVHSEDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLL
55.7
66.2
80–90
ECD_FGFR4b-Fc
SWVFLFFLSVTTGVHSDSLTSSNDDEDPKSHRDPSNRHSYPQQAPYWTHPQRMEKK
56.2
65.6
80–90
Secretion signal peptide sequence is underlined.
The proteins were purified from the culture supernatant using a single-step affinity chromatography on protein A Sepharose as described in Materials and Methods. SDS–PAGE analysis of the purification process was carried out, followed by Coomassie blue staining (Fig. 5A) and Western blotting (Fig. 5B). With optimized transient transfection and purification procedures we obtained high yields of all ECD_FGFRs-Fc proteins (Table 4) that were over 95% pure (Fig. 5A, B, D). The SDS–PAGE analysis of purified ECD_FGFRa-Fc proteins showed protein bands corresponding to about 100–110 kDa (Fig. 5D). The FGF receptors lacking D1 domain (ECD_FGFRb-Fc) migrated as an 80–90-kDa band in SDS–PAGE (Fig. 5D). All the purified ECD_FGFRs-Fc proteins gave unique bands on Western blots with anti-Fc antibodies (Fig. 5E), and the ECD_FGFRa-Fc proteins were additionally analyzed using antiECD_FGFR antibodies specific to the N-terminal region of FGFR, producing unique bands (Fig. 5F). As isolated, all the ECD_FGFR-Fc proteins were dimeric, probably owing to a disulphide bond in the hinge domain of the Fc, as evidenced by their migration upon SDS–PAGE under non-reducing conditions as a band of 190–230 kDa (Fig. 5C). Upon reduction they migrated slightly above their calculated monomeric molecular mass (100–110 kDa, Fig. 5C). Deglycosylation of ECD_FGFRs-Fc The apparent molecular weights of the recombinant proteins as determined by SDS–PAGE were higher than the calculated ones based on the amino acid sequence, and the band in the gel had a characteristic fuzzy appearance, suggesting glycosylation. (Fig. 5D, Table 4). We therefore investigated the N-linked glycosylation of the proteins by treating them with an N-glycosidase PNGase F. That treatment produced ECD_FGFRs-Fc molecules migrating faster and as a narrower band than the corresponding non-treated proteins (Fig. 6) – both features clearly indicating that originally the proteins were heavily modified by N-glycosylation. This
strongly suggests that the N-linked carbohydrates were responsible for the increase in the apparent mass observed on SDS–PAGE gel by more than 30 kDa. Mass spectrometry analysis The molecular weights of the recombinant proteins in their native (glycosylated) but reduced (monomeric) forms determined using MALDI-TOF/TOF mass spectrometry are given in Table 5. According to our SDS–PAGE analysis, approximately 35% of the ECD_FGFR-Fc protein molecular masses were attributed to glycans, however, mass spectrometry analysis showed that the carbohydrates constituted 13–18% of the mass of the glycoproteins (Table 5). When the proteins were first deglycosylated as described earlier, their MWs determined by mass spectrometry corresponded well to the calculated ones (data not shown). The N-terminal sequences of all the proteins were confirmed using MALDI-PSD-MS analysis (Table 5). The secretion signal peptide without the first methionine was present in all the mature secreted proteins. Conclusions In this study, we cloned, expressed and purified from CHO cells the extracellular domains of different types of FGFRs in fusion with the Fc antibody fragment. We believe that such recombinant proteins can conveniently be used as molecular targets for the selection of a range of FGFRs-specific antibodies. The Fc fragment allowed an efficient single-step purification of the proteins by affinity chromatography on Protein A Sepharose and increased the protein stability. Consistent with earlier data [23], the extracellular domains of FGFRs were glycosylated when expressed in the mammalian expression system. Our results demonstrate that the presented method enables one to produce fully glycosylated FGFR_ECD-Fc fusion proteins with high yield and purity. The
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glycosylation of the recombinant receptors indicates that the antibodies raised against the recombinant protein should efficiently recognize also native receptors. Acknowledgments We cordially thank Dr. Anna Cyranka-Czaja for her help and expertise. The work was supported by the National Science Centre, Poland (Grant 2011/02/A/NZ1/00066). References [1] S.K. Olsen, O.A. Ibrahimi, A. Raucci, F. Zhang, A.V. Eliseenkova, A. Yayon, C. Basilico, R.J. Linhardt, J. Schlessinger, M. Mohammadi, Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity, Proc. Natl. Acad. Sci. USA 101 (2004) 935–940. [2] M. Zakrzewska, E. Marcinkowska, A. Wiedlocha, FGF-1 – from biology, through engineering to potential medical applications, Crit. Rev. Clin. Lab. Sci. 45 (2008) 91–135. [3] D.E. Johnson, L.T. Williams, Structural and functional diversity in the FGF receptor multigene family, Adv. Cancer Res. 60 (1993) 1–41. [4] C.J. Powers, S.W. McLeskey, A. Wellstein, Fibroblast growth factors, their receptors and signaling, Endocr. Relat. Cancer 7 (2000) 165–197. [5] A. Wiedlocha, V. Sorensen, Signaling, internalization, and intracellular activity of fibroblast growth factor, Curr. Top. Microbiol. Immunol. 286 (2004) 45–79. [6] F. Wang, M. Kan, G. Yan, J. Xu, W.L. McKeehan, Alternately spliced NH2terminal immunoglobulin-like loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1, J. Biol. Chem. 270 (1995) 10231–10235. [7] E.M. Haugsten, A. Wiedlocha, S. Olsnes, J. Wesche, Roles of fibroblast growth factor receptors in carcinogenesis, Mol. Cancer Res. 8 (2010) 1439–1452. [8] J. Wesche, K. Haglund, E.M. Haugsten, Fibroblast growth factors and their receptors in cancer, Biochem. J. 437 (2011) 199–213. [9] V. Knights, S.J. Cook, De-regulated FGF receptors as therapeutic targets in cancer, Pharmacol. Ther. 125 (2010) 105–117. [10] V. Gelsi-Boyer, B. Orsetti, N. Cervera, P. Finetti, F. Sircoulomb, C. Rouge, L. Lasorsa, A. Letessier, C. Ginestier, F. Monville, et al., Comprehensive profiling of 8p11-12 amplification in breast cancer, Mol. Cancer Res. 3 (2005) 655–667. [11] K. Chin, S. DeVries, J. Fridlyand, P.T. Spellman, R. Roydasgupta, W.L. Kuo, A. Lapuk, R.M. Neve, Z. Qian, T. Ryder, et al., Genomic and transcriptional
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells Aleksandra Sokolowska-Wedzina a,1, Aleksandra Borek a,1, Julia Chudzian a, Piotr Jakimowicz b, Malgorzata Zakrzewska a, Jacek Otlewski a,⇑ a b
Faculty of Biotechnology, Department of Protein Engineering, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland Faculty of Biotechnology, Department of Protein Biotechnology, University of Wroclaw, Joliot-Curie 14a, 50-383 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Received 8 January 2014 and in revised form 26 March 2014 Available online 13 April 2014 Keywords: FGFR CHO Protein expression Glycosylation
a b s t r a c t The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2bFc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Fibroblast growth factor (FGF)2 signaling regulates numerous important biological processes including embryonic development, tissue homeostasis, angiogenesis and general metabolism. The biological effects of FGFs observed in target cells are mediated through specific cell-surface tyrosine kinase receptors, called fibroblast growth factor receptors (FGFRs) [1,2]. Four FGF receptors have been identified to date, FGFR-1 through FGFR-4, which share between 55% and 72% homology at the amino acid sequence level [3]. The prototypical FGFR consists of an extracellular ligand-binding region composed of three Ig-like domains (D1, D2, D3), a unique acidic serine-rich region (acid box), a transmembrane domain, and a well conserved split tyrosine kinase domain responsible for the activation of downstream signaling and biological responses [4,5]. ⇑ Corresponding author. Tel.: +48 71 375 2824. E-mail address: [email protected] (J. Otlewski). Equally contributing authors. 2 Abbreviations used: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; ECD, extracellular domain; SSP, secretion signal peptide; NSP, native signal peptide; PCR, polymerase chain reaction; PEI, polyethyleneimine; PBS, phosphate buffered saline. 1
http://dx.doi.org/10.1016/j.pep.2014.03.012 1046-5928/Ó 2014 Elsevier Inc. All rights reserved.
The first Ig-like domain (D1) and the acid box are involved in the auto-inhibitory function of the receptors, whereas the second and third Ig domains are critical for the ligand binding and its specificity. Regulation of the ligand-binding specificity of FGFR is essential for the control of FGF signaling and is primarily achieved by alternative splicing of FGFR1-3 transcripts [6]. An alternative splicing event in FGFR1-3 involving the exon encoding the C-terminal region of D3 results in two receptor isoforms (IIIb and IIIc) of different ligand-binding specificities. Additional alternative splicing events result in FGFR isoforms that lack D1 (in FGFR1 and 2), D1 and the D1–D2 linker (in FGFR2), or the D1–D2 linker (in FGFR3) [4]. Overexpression of FGFRs has been reported in a wide variety of tumors, such as breast, bladder, prostate, endometrial, lung and hematologic cancers, and has been associated with tumor progression and poor patient prognosis [7–9]. Depending on the localization and cancer type, different FGFRs are overexpressed. For example, elevated levels of FGFR1 [8,10–12], FGFR2 [13], and FGFR4 [14] are found in breast cancers, of FGFR2 in gastric cancers [15], overexpression of FGFR1 and FGFR3 is associated with papillary thyroid carcinoma [16,17], and of FGFR1 and FGFR4 with prostate cancers [18]. Moreover, low levels of FGFR are found on noncancer cells in the tumor vicinity. Therefore, FGFRs represent
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A. Sokolowska-Wedzina et al. / Protein Expression and Purification 99 (2014) 50–57 Table 1 Constructed plasmids encoding extracellular domain of FGFRs fused with Fc fragment. Plasmid name
Plasmid size
Amino acids of FGFRs
D3 domain splicing variant
Gene ID
RefSeq number (mRNA)
RefSeq number (protein)
pLEV113-ECD_FGFR1a-Fc pLEV113-ECD_FGFR2a-Fc pLEV113-ECD_FGFR3a-Fc pLEV113-ECD_FGFR4a-Fc pLEV113-ECD_FGFR1b-Fc pLEV113-ECD_FGFR2b-Fc pLEV113-ECD_FGFR3b-Fc pLEV113-ECD_FGFR4b-Fc
6924 bp 6936 bp 6927 bp 6912 bp 6656 bp 6669 bp 6618 bp 6621 bp
22–374 22–377 23–375 22–369 22–24, 120–374 22–36, 126–377 23, 124–375 119–369
IIIc IIIc IIIc – IIIc IIIc IIIc –
2260 2263 2261 2264 2260 2263 2261 2264
NM_015850.3 NM_000141.4 NM_000142.4 NM_002011.3 NM_015850.3 NM_000141.4 NM_000142.4 NM_002011.3
NP_056934.2 NP_000132.3 NP_000133.1 NP_002002.3 NP_056934.2 NP_000132.3 NP_000133.1 NP_002002.3
‘‘a’’ in the name of construct indicates whole extracellular domain of FGFR (containing D1, acid box, D2, D3), ‘‘b’’ indicates shortened fragment of extracellular domain, lacking D1 (acid box, D2, D3). ECD – extracellular domain.
an attractive target for anti-tumor therapy. Until now, small-molecule tyrosine kinase inhibitors and several antibodies have been tested to target FGFRs in various types of cancer [8,9,19]. In this study, we have cloned, expressed and purified variants of Fc-tagged extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2bFc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGF R4b-Fc, Table 1). Such fusion proteins are convenient molecular targets for the selection of a range of FGFR-specific antibodies. Materials and methods Construction of ECD_FGFR-Fc expression plasmids An expression construct ECD_FGFR1a-Fc was prepared in the mammalian expression vector pLEV113 (pLEV113-ECD_FGFR1aFc) by LakePharma (Belmont, CA). It contained a DNA fragment corresponding to the extracellular domain (ECD) of FGFR1 isoform IIIc (residues 22–374), which was preceded by a secretion signal peptide and followed by the sequence of human IgG1 Fc domain, with a nine-amino acid glycine-rich linker between the ECD-FGFR1 and Fc. cDNAs encoding full-length sequences of FGFR2, FGFR3 and FGFR4 were kindly provided by Antoni Wiedlocha’s Laboratory (Oslo University Hospital). These cDNAs together with pLEV 113-ECD_FGFR1a-Fc were then used to prepare expression constructs: pLEV113-ECD_FGFR1b-Fc, pLEV113-ECD_FGFR2a-Fc, pLEV 113-ECD_FGFR2b-Fc, pLEV113-ECD_FGFR3a-Fc, pLEV1113-ECD_ FGFR3b-Fc, pLEV113-ECD_FGFR4a-Fc, pLEV113-ECD_FGFR4b-Fc
(Table 1). All PCR primers used for plasmid preparation are summarized in Tables 2 and 3. The plasmids were verified by sequencing, amplified and purified using Plasmid Maxi Kit (Qiagen). Cell culture and transfection CHO-S cells (Invitrogen), adapted for growth in suspension, were cultured in serum- and protein-free PowerCHO-2CD medium (Lonza) supplemented with 8 mM L-glutamine and 1 penicillin/streptomycin solution. Cells were grown at 37 °C in a shaking incubator (140 rpm) with 8% CO2. Routine subculturing was conducted every 2–3 days at seeding density of 0.1– 0.3 106 cells/ml. One day prior to transfection, cells were seeded at a density of 0.6 106 cells/ml in culture medium. On the day of transfection CHO-S cells were centrifuged and cell pellet was resuspended in serum- and protein-free ProCHO4 medium at 2 106 cells/ml. Plasmid (1 mg/ml) and PEI (1 mg/ml) stocks were diluted separately in 150 mM NaCl, mixed at a 1:4 (w:w) ratio, incubated at RT for 10 min, and added to the cell culture (1.25 lg DNA per 1 106 cells, total volume added was 1/10 of the volume of transfected culture). Cells were incubated for 4 h in standard conditions (37 °C, 140 rpm, 8% CO2). After this time the culture was diluted with an equal volume of PowerCHO-2CD supplemented with 8 mM L-glutamine and 2 penicillin/streptomycin solution to obtain the cell density of about 1 106 cells/ml, and incubated at 31 °C. At day 2 following transfection the culture was supplemented with 4 mM L-glutamine and finally harvested at day 6.
Table 2 Primers used for preparation of pLEV113-ECD_FGFRa-Fc plasmids. Symbol
Name
A
FGFR1a-Kpn2I-Fc-F
5 CCTGTACCTGGAGTCCGGAGGAGGTGGTGCAGG 3
FGFR1a-Kpn2I-Fc-R
50 CCTGCACCACCTCCTCCGGACTCCAGGTACAGG 30
MutFGFR2-F
50 GTGAATGTCACAGATGCCATCTCATCAGGTGATGATGAGGAT GACACCGATGG 30
MutFGFR2-R
50 CCATCGGTGTCATCCTCATCATCACCTGATGAGATGGCATCT GTGACATTCAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCGGCCCT CCTTCAGTTTAGTTGAGG 30
B
C
FGFR2a-SSP-F
D
FGFR2a-Kpn2I-R
E
FGFR3a-SSP-F
F
FGFR3a-Kpn2I-R
G
Sequence 0
Specification 0
Introduction of Kpn2I restriction site Kpn2I restriction site removal
Addition of SSP fragment
50 tacgTCCGGACTCCAGGTAGTCTGGGGAAGC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGAGTCCTT GGGGACGGAGCAGC 30
Addition of Kpn2I restriction site
50 tacgTCCGGAGCCTGCATACACACTGCCCGCC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCTGGAGGC CTCTGAGGAAGTGG 30
Addition of Kpn2I restriction site
FGFR4a-SSP-F
H
FGFR4a-Kpn2I-R
50 tacgTCCGGAGTCCGTATACCTGGCCTCGGG 30
Addition of Kpn2I restriction site
I
HindIII-SSP-F
50 ctccAAGCTTTGAACCACCATGGAATGGAGCTGGGTCTTTCTCTT CTTCCTGTCAGTAACGACTGG 30
Addition of C-terminal fragment of SSP with HindIII restriction site
SSP – secretion signal peptide.
Addition of SSP fragment
Addition of SSP fragment
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Table 3 Primers used for preparation of pLEV113-ECD_FGFRb-Fc constructs. Symbol J K L M N O P R S T
Name FGFR1b-F FGFR1bSSP-F FGFR2b-1-F FGFR2b-2-F FGFR2bSSP-F FGFR3b-F FGFR3bSSP-F FGFR4b-F FGFR4bSSP-F Fc-Not1-R
Sequence 0
Specification 0
5 GATGCTCTCCCCTCCTCGGAGGATGATG 3 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCAGGCCGTCCGATGCTCTCCCCTCCTCGGAGGATGATG 30 50 GATGCCATCTCATCAGGTGATGATGAGGATGACAC 30 50 CTTCAGTTTAGTTGAGGATACCACATTAGAGCCAGAAGATGCCATCTCATCAGGTGATGATGAGGATGACAC 30 50 TTCTTCCTGTCAGTAACGACTGGTGTCCACTCCCGGCCCTCCTTCAGTTTAGTTGAGGATACCACATTAGAGCC 30 50 GACGCTCCATCCTCGGGAGATGACGAAGAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGAGGACGCTCCATCCTCGGGAGATGACGAAGAC 30
Cloning D2D3-Fc fragment Addition of SSP fragment
50 GACTCCTTGACCTCCAGCAACGATGATGAGGAC 30 50 CTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCCGACTCCTTGACCTCCAGCAACGATGATGAGGAC 30
Cloning D2D3-Fc fragment Addition of SSP fragment
50 tcgaGCGGCCGCTCATTTACCCGGAGAC 30
Cloning of ECD-FGFR-Fc
Cloning D2D3-Fc fragment Addition of first 15 amino acids of FGFR2 Addition of SSP fragment Cloning D2D3-Fc fragment Addition of SSP fragment
SSP – secretion signal peptide.
Fig. 1. Construction of pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc plasmids. SSP – secretion signal peptide, L – linker, NSP – native signal peptide.
Purification of FGFRs-Fc Cell culture was harvested by centrifugation at 15,000 g for 20 min. The resultant supernatant was filtered through 0.22micron filter units and loaded onto a Protein A Sepharose column (GE Healthcare) pre-equilibrated with PBS. The unbound fraction was removed and the resin was washed first with 500 ml of washing buffer A: 30 mM Na2HPO4, 20 mM NaH2PO4, 350 mM NaCl, 0.1% Tween 20, 2 mM EDTA, pH 7.4 and then with 500 ml of washing buffer B: 30 mM Na2HPO4, 20 mM NaH2PO4, 650 mM NaCl, 2 mM EDTA, pH 7.4. Proteins specifically bound to the resin were
eluted with 100 mM triethylamine pH 11.5, and then immediately neutralized with 1 M Tris–HCl pH 7.2. Fractions with the highest protein concentrations were collected, dialyzed against PBS, and concentrated using Centriprep 10 K centrifugal filter units (Millipore). Protein purity was checked by SDS–PAGE and their identity was confirmed by mass spectrometry. Deglycosylation of ECD_FGFRs-Fc The recombinant proteins were treated with PNGase F according to the manufacturer’s instructions (New England BioLabs). In
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brief, 2 ll of 10 glycoprotein denaturing buffer was added to 2.5 lg of protein in 20 ll buffer and incubated at 95 °C for 10 min, then 3 ll of 10xG7 reaction buffer, 3 ll of NP-40 and 1 ll (500 U/ll) of PNGase F were added to each reaction mix and samples were incubated at 37 °C for 1 h. The effect of the deglycosylation on the molecular mass of the proteins was determined by SDS–PAGE and mass spectrometry.
co-crystallization. Whole-protein spectra were recorded in the high mass range with a 4800 Plus MALDI TOF/TOF (AB SCIEX) mass spectrometer. Next, N-terminal sequencing was done using MALDI-PDS-MS analysis for peptide mass range of 600–4000 Da.
SDS–PAGE and Western blotting
Construction of ECD_FGFR-Fc expression plasmids
The proteins were separated on SDS–PAGE 4–20% gradient gel (Pierce, Thermo Scientific) and either stained with Coomassie blue or electrotransferred to a nitrocellulose membrane (Millipore). The membrane was incubated with the anti-FGFRs antibodies (Flg (S-16), Santa Cruz, sc-31169; Bek (H-80), Santa Cruz, sc-20735; FGFR3 (ZZ10), Santa Cruz, sc-73994; FGFR4 (A10), Santa Cruz, sc136988) and anti-Fc-HRP-conjugated antibody (KPL, 4-10-2020) in 1% BSA for 1.5 h at RT. After incubation with the primary antibodies the membrane was washed with 0.1% Tween 20 in PBS and incubated with the appropriate HRP-conjugated secondary antibodies in 1% BSA for 1 h at RT and finally washed with 0.1% Tween 20 in PBS. The blot was developed with the ECL reagent (Pierce, Thermo Scientific) according to the manufacturer’s instructions.
For expression of recombinant ECD_FGFR-Fc fusion proteins, plasmids pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc were constructed. The cloning procedure is presented in Fig. 1. Plasmid pLEV113-ECD_FGFR1a-Fc was modified by introduction of a Kpn2I restriction site (TCCGGA) in the linker region between the extracellular domain of FGFR1a and Fc using the pair of primers marked A (Table 2). The resulting plasmid was digested with HindIII/Kpn2I and named pLEV113-Kpn2I-Fc (Fig. 1A). Simultaneously, cDNAs encoding FGFR2 residues 22– 377, FGFR3 residues 23–375 or FGFR4 residues 22–369 were amplified by two rounds of PCR. The first PCR introduced at the 50 end a fragment encoding part of the extracellular targeting signal peptide (primers C, E, G, Table 2) and a Kpn2I site at the 30 end (primers D, F, H, Table 2). The second PCR completed the secretion signal peptide and introduced a HindIII site (primer I and D, F, H) (Fig. 1B). Additionally, FGFR2 cDNA was mutated to remove its own Kpn2I site (TCCGGA into TCAGGT) without changing amino acid sequence (pair of primers B, Table 2). Finally, the PCR products were digested and ligated into the HindIII/Kpn2I sites of the modified pLEV113-Kpn2I-Fc expression vector (Fig. 1C and 2). The
Mass spectrometry Protein samples were diluted with MALDI matrix (sinapinic acid 10 mg/ml in 50% acetonitrile, 0.1% TFA) at a ratio 1:2, 1:4 or 1:8 and deposited onto an Opti-TOF™ 384 Well Plate (AB SCIEX) for
Results and discussion
Fig. 2. Map of vector pLEV113 with ECD_FGFRs-Fc cloning site indicated. SSP – secretion signal peptide.
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Fig. 3. Cloning procedure for construction of pLEV113-ECD_FGFR1b-Fc, pLEV113-ECD_FGFR2b-Fc, pLEV113-ECD_FGFR3b-Fc, and pLEV113-ECD_FGFR4b-Fc plasmids. Sequence of FGFR2 D1 to be removed was determined according to naturally occurring variant of FGFR2 lacking D1 (RefSeq Number: NP_001138391.1, Isoform IIIb). SSP – secretion signal peptide, L – Linker.
sequences of recombinant plasmids pLEV113-ECD_FGFR2a-Fc, pLEV113-ECD_FGFR3a-Fc and pLEV113-ECD_FGFR4a-Fc were confirmed by sequencing (LGC Genomics). For expression of respective proteins lacking the D1 domain, plasmids pLEV113-FGFR1b-Fc, pLEV113-FGFR2b-Fc, pLEV113FGFR3b-Fc, and pLEV113-FGFR4b-Fc were constructed. Their cloning procedure is presented in Fig. 3. DNA fragments corresponding to FGFR1 residues 22–24 and 120–374; FGFR2 residues 22–36 and 126–377; FGFR3 residues 23 and 124–375, and FGFR4 residues 119–369 were amplified with the downstream Fc sequence from
pLEV113-ECD_FGFRa-Fc plasmids by three rounds of PCR with one additional round for the ECD_FGFR2b-Fc construct. DNA sequences corresponding to the above fragments of all four constructs were amplified by the first PCR (primers J, L, O, R and T, Table 3). The second PCR introduced a fragment encoding the Nterminal part of the extracellular targeting signal peptide (primers K, P, S, and T, Table 3) of FGFR1b-Fc, FGFR3b-Fc, FGFR4b-Fc, and the first 15 amino acids of FGFR2b-Fc (primers M and T, Table 3). The additional PCR introduced a fragment encoding the N-terminal part of extracellular targeting signal peptide of FGFR2b-Fc (primers
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Fig. 4. Expression of recombinant ECD_FGFR-Fc proteins. As an example, expression of ECD_FGFR1a-Fc is presented. Culture supernatant was analyzed at each day of expression by (A) SDS–PAGE under reducing conditions followed by Coomassie blue staining and (B) Western blotting with anti-FGFR1 antibodies. (C) Cell growth and recombinant protein production by CHO-S cells. Histograms represent recombinant protein production expressed as band areas on anti-FGFR1 Western blots. The line represents cell growth expressed as viable cells/ml of culture. Additional glutamine supplementation on day 2 of protein expression is marked. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Purification of recombinant ECD_FGFR-Fc proteins. Purification steps were analyzed by (A) SDS–PAGE under reducing condition followed by Coomassie blue staining and (B) Western blotting with anti-FGFR4 antibodies. (C) Recombinant ECD-FGFR-Fc protein is isolated as a dimer. Purified ECD_FGFR4a-Fc was analyzed under non-reducing (sample buffer without b-mercaptoethanol and no heating) or reducing (sample buffer supplemented with b-mercaptoethanol, sample heated at 99 °C for 10 min) conditions, followed by Coomassie blue staining. Analysis of all purified recombinant ECD_FGFR-Fc proteins by (D) SDS–PAGE under reducing conditions followed by Coomassie blue staining, (E) Western blotting with anti-Fc antibodies, (F) Western blotting with specific anti-FGFR antibodies. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
N and T, Table 3). The last PCR completed the secretion signal peptide and introduced a HindIII site to all constructs (primers I and T, Table 3) (Fig. 3A). Agarose gel extraction was performed after each round of PCR. Final PCR products were ligated into the HindIII/NotI sites of the pLEV113 vector (Fig. 3B and C). The sequences of pLEV113-FGFR1b-Fc, pLEV113-FGFR2b-Fc, pLEV113-FGFR3b-Fc, and pLEV113-FGFR4b-Fc plasmids were confirmed as above. Expression and purification of recombinant FGF receptors A mammalian cell transient transfection system was used to express fully glycosylated ECD_FGFR-Fc fusion proteins. The use of serum-free medium reduced the level of extraneous proteins in the culture medium and facilitated the detection and purification of secreted recombinant proteins [20–22]. The level of the recombinant proteins in culture medium during expression was monitored and analyzed by SDS gel electrophoresis in reducing conditions followed by Coomassie blue staining (Fig. 4A) and by
Table 4 Yields of individual ECD_FGFR-Fc protein preparations. Recombinant protein
Average protein yield [mg protein/l culture] ± SD (n = 3)
ECD_FGFR1a-Fc ECD_FGFR2a-Fc ECD_FGFR3a-Fc ECD_FGFR4a-Fc ECD_FGFR1b-Fc ECD_FGFR2b-Fc ECD_FGFR3b-Fc ECD_FGFR4b-Fc
20.0 ± 3.6 21.1 ± 1.8 18.3 ± 5.2 21.2 ± 2.7 12.7 ± 2.7 14.1 ± 4.1 14.8 ± 2.9 13.2 ± 4.3
Western blot analysis using specific anti-FGFR antibodies (Fig. 4B). Fig. 4C summarizes the time-course of cell growth and recombinant protein production in CHO-S cells for 6 days following transfection.
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Fig. 6. SDS–PAGE of native and deglycosylated recombinant ECD_FGFR-Fc proteins. Purified ECD_FGFR-Fc proteins were subjected to electrophoresis under denaturating conditions without or with prior PNGase F treatment.
Table 5 Characteristics of ECD_FGFR-Fc proteins. MS MW of proteins [kDa] (monomer)
SDS–PAGE MW of proteins [kDa] (monomer)
67.4
80.0
100–110
67.9
78.2
100–110
WSWVFLFFLSVTTGVHSESLGTEQRVVGRAAEVPGPEPGQQEQLVFGSGDAVELSCPP
66.5
78.5
100–110
ECD_FGFR4a-Fc
WSWVFLFFLSVTTGVHSLEASEEVELEPCLAPSLEQQEQELTVALGQPVRLCCGRA
66.9
79.3
100–110
ECD_FGFR1b-Fc
EWSWVFLFFLSVTTGVHSRPSDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSP
57.1
69.7
80–90
Recombinant protein
N-terminal sequence determined using MALDI-PSD-MS analysis
Calculated MW [kDa] (monomer)
ECD_FGFR1a-Fc
SWVFLFFLSVTTGVHSRPSPTLPEQAQPWGAPVEVESFLVHPGDLLQL
ECD_FGFR2a-Fc
EWSWVFLFFLSVTTGVHSRPSFSLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEVR
ECD_FGFR3a-Fc
ECD_FGFR2b-Fc
SWVFLFFLSVTTGVHSRPSFSLVEDTTLEPEDAISSGDDEDDTDGAEDFVSENSNN
58.1
68.4
80–90
ECD_FGFR3b-Fc
SWVFLFFLSVTTGVHSEDAPSSGDDEDGEDEAEDTGVDTGAPYWTRPERMDKKLL
55.7
66.2
80–90
ECD_FGFR4b-Fc
SWVFLFFLSVTTGVHSDSLTSSNDDEDPKSHRDPSNRHSYPQQAPYWTHPQRMEKK
56.2
65.6
80–90
Secretion signal peptide sequence is underlined.
The proteins were purified from the culture supernatant using a single-step affinity chromatography on protein A Sepharose as described in Materials and Methods. SDS–PAGE analysis of the purification process was carried out, followed by Coomassie blue staining (Fig. 5A) and Western blotting (Fig. 5B). With optimized transient transfection and purification procedures we obtained high yields of all ECD_FGFRs-Fc proteins (Table 4) that were over 95% pure (Fig. 5A, B, D). The SDS–PAGE analysis of purified ECD_FGFRa-Fc proteins showed protein bands corresponding to about 100–110 kDa (Fig. 5D). The FGF receptors lacking D1 domain (ECD_FGFRb-Fc) migrated as an 80–90-kDa band in SDS–PAGE (Fig. 5D). All the purified ECD_FGFRs-Fc proteins gave unique bands on Western blots with anti-Fc antibodies (Fig. 5E), and the ECD_FGFRa-Fc proteins were additionally analyzed using antiECD_FGFR antibodies specific to the N-terminal region of FGFR, producing unique bands (Fig. 5F). As isolated, all the ECD_FGFR-Fc proteins were dimeric, probably owing to a disulphide bond in the hinge domain of the Fc, as evidenced by their migration upon SDS–PAGE under non-reducing conditions as a band of 190–230 kDa (Fig. 5C). Upon reduction they migrated slightly above their calculated monomeric molecular mass (100–110 kDa, Fig. 5C). Deglycosylation of ECD_FGFRs-Fc The apparent molecular weights of the recombinant proteins as determined by SDS–PAGE were higher than the calculated ones based on the amino acid sequence, and the band in the gel had a characteristic fuzzy appearance, suggesting glycosylation. (Fig. 5D, Table 4). We therefore investigated the N-linked glycosylation of the proteins by treating them with an N-glycosidase PNGase F. That treatment produced ECD_FGFRs-Fc molecules migrating faster and as a narrower band than the corresponding non-treated proteins (Fig. 6) – both features clearly indicating that originally the proteins were heavily modified by N-glycosylation. This
strongly suggests that the N-linked carbohydrates were responsible for the increase in the apparent mass observed on SDS–PAGE gel by more than 30 kDa. Mass spectrometry analysis The molecular weights of the recombinant proteins in their native (glycosylated) but reduced (monomeric) forms determined using MALDI-TOF/TOF mass spectrometry are given in Table 5. According to our SDS–PAGE analysis, approximately 35% of the ECD_FGFR-Fc protein molecular masses were attributed to glycans, however, mass spectrometry analysis showed that the carbohydrates constituted 13–18% of the mass of the glycoproteins (Table 5). When the proteins were first deglycosylated as described earlier, their MWs determined by mass spectrometry corresponded well to the calculated ones (data not shown). The N-terminal sequences of all the proteins were confirmed using MALDI-PSD-MS analysis (Table 5). The secretion signal peptide without the first methionine was present in all the mature secreted proteins. Conclusions In this study, we cloned, expressed and purified from CHO cells the extracellular domains of different types of FGFRs in fusion with the Fc antibody fragment. We believe that such recombinant proteins can conveniently be used as molecular targets for the selection of a range of FGFRs-specific antibodies. The Fc fragment allowed an efficient single-step purification of the proteins by affinity chromatography on Protein A Sepharose and increased the protein stability. Consistent with earlier data [23], the extracellular domains of FGFRs were glycosylated when expressed in the mammalian expression system. Our results demonstrate that the presented method enables one to produce fully glycosylated FGFR_ECD-Fc fusion proteins with high yield and purity. The
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glycosylation of the recombinant receptors indicates that the antibodies raised against the recombinant protein should efficiently recognize also native receptors. Acknowledgments We cordially thank Dr. Anna Cyranka-Czaja for her help and expertise. The work was supported by the National Science Centre, Poland (Grant 2011/02/A/NZ1/00066). References [1] S.K. Olsen, O.A. Ibrahimi, A. Raucci, F. Zhang, A.V. Eliseenkova, A. Yayon, C. Basilico, R.J. Linhardt, J. Schlessinger, M. Mohammadi, Insights into the molecular basis for fibroblast growth factor receptor autoinhibition and ligand-binding promiscuity, Proc. Natl. Acad. Sci. USA 101 (2004) 935–940. [2] M. Zakrzewska, E. Marcinkowska, A. Wiedlocha, FGF-1 – from biology, through engineering to potential medical applications, Crit. Rev. Clin. Lab. Sci. 45 (2008) 91–135. [3] D.E. Johnson, L.T. Williams, Structural and functional diversity in the FGF receptor multigene family, Adv. Cancer Res. 60 (1993) 1–41. [4] C.J. Powers, S.W. McLeskey, A. Wellstein, Fibroblast growth factors, their receptors and signaling, Endocr. Relat. Cancer 7 (2000) 165–197. [5] A. Wiedlocha, V. Sorensen, Signaling, internalization, and intracellular activity of fibroblast growth factor, Curr. Top. Microbiol. Immunol. 286 (2004) 45–79. [6] F. Wang, M. Kan, G. Yan, J. Xu, W.L. McKeehan, Alternately spliced NH2terminal immunoglobulin-like loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1, J. Biol. Chem. 270 (1995) 10231–10235. [7] E.M. Haugsten, A. Wiedlocha, S. Olsnes, J. Wesche, Roles of fibroblast growth factor receptors in carcinogenesis, Mol. Cancer Res. 8 (2010) 1439–1452. [8] J. Wesche, K. Haglund, E.M. Haugsten, Fibroblast growth factors and their receptors in cancer, Biochem. J. 437 (2011) 199–213. [9] V. Knights, S.J. Cook, De-regulated FGF receptors as therapeutic targets in cancer, Pharmacol. Ther. 125 (2010) 105–117. [10] V. Gelsi-Boyer, B. Orsetti, N. Cervera, P. Finetti, F. Sircoulomb, C. Rouge, L. Lasorsa, A. Letessier, C. Ginestier, F. Monville, et al., Comprehensive profiling of 8p11-12 amplification in breast cancer, Mol. Cancer Res. 3 (2005) 655–667. [11] K. Chin, S. DeVries, J. Fridlyand, P.T. Spellman, R. Roydasgupta, W.L. Kuo, A. Lapuk, R.M. Neve, Z. Qian, T. Ryder, et al., Genomic and transcriptional
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