Accepted Manuscript Expression, purification and characterization of galectin-1 in Escherichia coli Zhen Shu, Jing Li, Nan Mu, Yuan Gao, Tonglie Huang, Ying Zhang, Zenglu Wang, Meng Li, Qiang Hao, Weina Li, Liqing He, Cun Zhang, Wei Zhang, Xiaochang Xue, Yingqi Zhang PII: DOI: Reference:

S1046-5928(14)00078-3 http://dx.doi.org/10.1016/j.pep.2014.03.013 YPREP 4491

To appear in:

Protein Expression and Purification

Received Date: Revised Date:

21 February 2014 31 March 2014

Please cite this article as: Z. Shu, J. Li, N. Mu, Y. Gao, T. Huang, Y. Zhang, Z. Wang, M. Li, Q. Hao, W. Li, L. He, C. Zhang, W. Zhang, X. Xue, Y. Zhang, Expression, purification and characterization of galectin-1 in Escherichia coli, Protein Expression and Purification (2014), doi: http://dx.doi.org/10.1016/j.pep.2014.03.013

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Expression, purification and characterization of galectin-1 in Escherichia coli Zhen Shua,#, Jing Lia,b,#, Nan Mua, Yuan Gao a, Tonglie Huanga,c, Ying Zhanga, Zenglu Wanga, Meng Lia, Qiang Hao a, Weina Lia, Liqing Hea, Cun Zhanga, Wei Zhanga, Xiaochang Xuea,*, Yingqi Zhanga,* a

State Key Laboratory of Cancer Biology, Department of Biopharmaceutics, School of

Pharmacy, Fourth Military Medical University, Xi’an 710032, China b

c

Current address: Baoji Municipal Central Blood Station, Baoji 721006, China

Key Laboratory of Biomedical Information Engineering of Ministry of Education, School

of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China AB STRACT As a member of beta-galactoside-binding proteins family, Galectin-1 (Gal-1) contains a single carbohydrate recognition domain, by means of which it can bind glycans both as a monomer and as a homodimer. Gal-1 is implicated in modulating cell-cell and cell-matrix interactions and may act as an autocrine negative growth factor that regulates cell proliferation. Besides, it can also suppress TH1 and TH17 cells by regulating dendritic cell differentiation or suppress inflammation via IL-10 and IL-27. In the present study, Gal-1 monomer and concatemer (Gal-1②), which can resemble Gal-1 homodimer, were #

These authors contributed equally to this work.

*Corresponding authors. Fax: +86 2983247213. E-mail addresses: [email protected], [email protected]. 1

Abbreviations used: Gal-1, Galectin-1; CRD, carbohydrate recognition domain; ConA, Concanavalin

A; LB, Luria-Bertani; IPTG, Isopropyl β-D-1-thiogalactopyranoside.

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expressed in Escherichia coli and their bioactivities were analyzed. The results of this indicate that both Gal-1and Gal-1② were expressed in Escherichia coli in soluble forms with a purity of over 95% after purifying with ion-exchange chromatography. Clearly, both Gal-1 and Gal-1② can effectively promote erythrocyte agglutination in hemagglutinating activity assays and inhibit Jurkat cell proliferation in MTT assays. All these data demonstrate that bacterially-expressed Gal-1 and Gal-1② have activities similar to those of wild type human Gal-1 whereas the bioactivity of concatemer Gal-1② was stronger than those of the bacterially-expressed and wild type human Gal. Keywords: Galectin-1; Escherichia coli; Soluble expression; Characterization; Erythrocyte agglutination Introduction As a member of β-galactoside-binding protein family implicated in modulating cell-cell and cell-matrix interactions [1, 2], galectins are composed of β-galactoside binding lectins containing homologous carbohydrate recognition domains (CRDs), which are highly conserved amino acid sequences in the 14 galectin family members that have been identified in mammals thus far [3]. According to their structural features, galectins have been classified into proto-, chimera- and tandem-repeat types [4]. Prototype galectins (Galectins-1, -2, -5, -7, -10, -11, -13, -14 and -15) contain one CRD domain, and usually form homodimers of non-covalently linked subunits [5]. In contrast, in addition to one CRD domain, chimera-type galectins (Galectins-3) have a non-carbohydrate-binding domain [6]. Tandem-repeat type galectins (Galectins-4, -6, -8, -9 and -12) are comprised of two different CRDs joined by a linker peptide and can cross-link glycoproteins due to 2

the presence of more than one CRDs [5]. Galectins of all the three types have hemagglutinating activities, which are attributable to their bivalent carbohydrate-binding properties [7]. Galectin-1 (Gal-1)1, a 135-amino acid protein in humans, is encoded by the LGALS1 gene which contains four exons [8, 9]. It is ubiquitously distributed in the nucleus, cytoplasm, cell surface and the extracellular space. Although it doesn’t have a traditional signal peptide sequence, it is still secreted across the plasma membrane by one or more unidentified, non-classical and secretory pathways [10]. Gal-1 appears both in the form of a monomer and a non-covalent homodimer that contains a single CRD domain, by means of which, Gal-1 can bind glycans. These two forms of Gal-1 are interconverted as homodimers disassociating spontaneously at low concentration [11]. Gal-1 may act as an autocrine negative growth factor that regulates proinflammatory lymphocyte adhesion, migration, polarization, proliferation, apoptosis and differentiation [12-14]. Accumulating evidence has shown that Gal-1 exerts its anti-inflammatory effect by inducing and differentiating CD4 +Foxp3 + regulatory T cells (Treg) and CD4 +IL-10 + T cells (Tr1) [15, 16], producing more anti-inflammatory cytokines interleukin (IL)-10 and IL-27 [12, 17, 18], and inducing innate immunce cells such as dendritic cells and macrophages towards a tolerance phenotype that suppresses differentiation of T helper 1 (TH1) and T helper 17 (TH17) cells [19-21]. As a result, Gal-1 is critically involved in various pathological states, including autoimmune diseases and tumor metastasis [22-25], and owing to its critical involvement in the states, recombinant Gal-1 has been used to control various autoimmune and chronic immune-pathological disorders [26, 27].

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Noorjahan Panjwani and his colleagues found that application of recombinant Gal-1 (rGal-1) after ocular P. aeruginosa infection significantly ameliorated corneal lesion severity by shifting pro-inflammatory TH17-mediated pathology toward anti-inflammatory TH2- and Tr1-type immune responses [20]. Liu et al. reported that recombinant Gal-1 has an anti-viral activity which is capable of reducing LCDV pathogenicity [4]. Although recombinant Gal-1 has been expressed in different systems and demonstrated to have potential anti-inflammatory activity, expression and identification of Gal-1 tandem repeats remain poorly characterized. Given that dimerization of prototype galectin is usually essential for the crosslinking of glycans and subsequent cellular signaling, the aim of this study was therefore to clone and identify the human Gal-1 monomer and concatemer (Gal-1②), which can mimic homodimer of Gal-1, and to compare the bioactivity of these recombinant Gal-1 proteins as well. Materials and methods Reagents Restriction enzymes, Phusion High-Fidelity DNA polymerase and buffers used for cloning were purchased from New England Biolabs (Ipswich, MA). T4 DNA ligase was from TaKaRa (Dalian, China). Oligonucleotides primers were synthesized by BioAsia (Beijing, China). The bacterial expression vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and Concanavalin A (ConA) were from Sigma-Aldrich (St. Louis, MO). Q-Sepharose Fast Flow and SP-Sepharose Fast Flow resins were purchased from GE

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Healthcare Life Sciences (Uppsala, Sweden). Mouse anti-human Gal-1 monoclonal antibody and HRP-conjugated rabbit anti-mouse IgG were from Abcam (UK, Cambridge). All other reagents were of analytical grade. Construction of recombinant plasmids pET-22b-Gal-1 and pET-22b-Gal-1② We have previously amplified gal-1 gene from human cDNA library with primers 1 and 2 listed in Table 1 and cloned it into pACT plasmid between BamH I and Kpn I restriction enzyme sites. DNA sequencing confirmed that the gene was correct (NCBI reference sequence: NM_002305.3) and the plasmid was named “pACTG”. For the construction of Gal-1 prokaryotic expression vector, the gal-1 gene was amplified from the pACTG plasmid with primers 3 and 4 (with Nde I and Sal I restriction sites respectively introduced and underlined). The PCR products were digested with Nde I and Sal I restriction enzymes, recovered from agarose gel and cloned into pET-22b(+) plasmid. pET-22b(+) contains a strong T7 promoter in front of multiple cloning sites (MCS) and thereby allows high level expression of recombinant genes located downstream. For the construction of Gal-1② expression vector, one copy of gal-1 gene was amplified from pACTG plasmid with primers 3 and 5 and Nde I and BamH I enzymes recognition sites were added to the products. Another copy was obtained with primers 4 and 6 and BamH I and Sal I enzyme recognition sites added. The two PCR products were digested and cloned into pET-22b(+) plasmid between Nde I and Sal I sites. All the recombinant plasmids were transformed into E. coli DH5α competent cells and plasmids were extracted and identified by DNA sequencing. The resulting correct plasmids were designated as “pET-G” and “pET-G②”. Finally E. coli strain BL21 (DE3), which can produce T7 5

RNA polymerase once induced by isopropyl β-D-thiogalactopyranoside (IPTG), was transformed with pET-G and pET-G② for recombinant gene expression. Table 1 Expression of Gal-1 monomer and Gal-1 ② concatemer A single colony was selected from pET-G and pET-G ② transformed plates respectively and inoculated in 5 ml of Luria-Bertani (LB) medium supplemented with 100 µg/ml of ampicillin and grown at 37°C with 200 rpm shaking overnight. The cultures were then transferred into 200 ml fresh medium in a shake flask. Protein expression was induced using 1 mM IPTG when the OD600 of the culture reached 0.6. Cells were harvested at 12,000 rpm for 20 min after 4 h of induction and the pellet was used for purification. One milliliter of the culture was collected and the pellet was resuspended in 100 µl ddH2O, mixed with 5 × SDS loading buffer, and heated at 95°C for 10 min. The sample was centrifuged at 12,000 rpm for 6 min and 10 µl supernatant was analyzed by SDS-PAGE. The uninduced sample was used as negative control. Fermentation was performed using a 5-L stirred Biostat® bioreactor as previously described [28]. In brief, a single colony was inoculated into 10 ml of LB medium and cultured at 37°C with shaking overnight. After that, 200 ml of semi-defined medium supplemented with ampicillin was inoculated with the overnight culture and grown at 37°C until the OD600 reached 2-3. Finally, 5 L sterile media (5 g/L Tryptone, 5 g/L yeast extract, 10 g/L glycerol, 2 g/L KH2PO4, 4 g/L K2HPO4, and 3 g/L MgSO4) were aseptically added to the fermentor and inoculated with the culture as described above. Fermentation was performed using the following parameters: 37°C, pH 7.0, airflow 5 L 6

pm (1 vvm), agitation 250-800 rpm, and dissolved oxygen 30%. When the OD600 reached 10.0, the cultures were induced by addition of IPTG (final concentration is 3 mM) for 4 h. Cells were harvested by centrifugation at 15,000 rpm for 25 min at 4°C. Purification of Gal-1 and Gal-1 ② To purify recombinant Gal-1 and Gal-1②, 10 g wet weight of cell pellets of BL21 (DE3) / pET-22b-Gal-1 or BL21 (DE3) / pET-22b-Gal-1② was suspended in 100 ml of lysis buffer (20 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) in a beaker on ice. The cells were disrupted by sonication and the supernatant was collected by centrifugation at 12,000 rpm for 20 min at 4°C. Then, the supernatant was thoroughly dialyzed against 30 times volume of buffer A (20 mM Tris-HCl pH 7.4, 1 mM EDTA) for 24 h and the insoluble particles were removed by filtering through a 0.22 µm syringe filter (VWR, West Chester, PA). A Q-Sepharose Fast Flow 26 × 200 mm column with 20 ml of column volume (CV) was equilibrated by buffer A with 5 CV, the samples were then loaded onto the column at a speed of 1 ml/min (ÄKTA purifier, GE Healthcare) at room temperature. The column was eluted with a linear gradient of NaCl from 0 to 1 M at a flow rate of 1 ml/min with 10 CV of buffer B (buffer A + 1.0 M NaCl, pH 7.4). Following that, the eluted fractions were collected and dialyzed against buffer C (20 mM citric acid- citrate, 1 mM EDTA, pH 4.0) thoroughly. The resulting solution was applied to a same size of SP-Sepharose Fast Flow column pre-equilibrated with buffer C and eluted with a 0-100% linear gradient of buffer D (buffer C + 1.0 M NaCl, pH 4.0) with 3.5 CV (for Gal-1) or 6.0 CV (for Gal-1②), respectively. The proteins were monitored by measuring the UV absorbance at 280 nm. The eluted fractions were diluted to 1 mg/ml and then pooled and 7

dialyzed against 20 mM phosphate buffer (pH 7.2) thrice at 4°C and stored at -20°C for later use. The purity of the recombinant proteins was determined by SDS-PAGE and HPLC. Western-blot analysis For Western blot, all the bacterial proteins were transferred to nitrocellulose membranes (0.22 µm; Invitrogen) after SDS-PAGE using a Bio-Rad Trans-Blot Semi-Dry electrophoretic cell. Western blot analyses were carried out using a mouse anti-human Gal-1 monoclonal antibody, followed by incubation with HRP-labeled goat anti-mouse IgG

antibody.

The

immunoreactive

proteins

were

visualized

with

enhanced

chemiluminescence (ECL) Western blotting substrate (Thermo Fisher Scientific, USA). Hemagglutinating activity assay The hemagglutinating activity of Gal-1 and Gal-1 ② was assayed according to methods reported previously and optimized [29]. In brief, blood samples from Balb/c mice were collected into heparinized tubes. Erythrocytes were pelleted by centrifugation at 1500 rpm for 10 min, washed three times with 0.9% sterilized sodium chloride saline (pH 7.0). Then, the cells were treated with 0.03% glutaraldehyde in PBS on ice for 1 h, followed by wash with PBS for three times. Finally, erythrocytes were resuspended in the saline at a concentration of 2% (v/v) and stored at 4°C for later use. Hemagglutination assays were performed in 96-well plates. Recombinant Gal-1 and Gal-1② at twofold serial dilutions (ranging from 1.6 to 200 µg/ml) in 50 µl of saline (pH 7.0) were incubated with 50 µl of 2% erythrocytes suspensions in triplicate for 2 h at room temperature, and

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each well was examined visually with unaided eyes for complete agglutination. Recombinant protein titer is reported as the reciprocal of the highest dilution that can cause detectable agglutination of erythrocytes. ConA and PBS were used as positive and negative controls, respectively. Cellular anti-proliferative activity of Gal-1 and Gal-1② The anti-proliferative activity of Gal-1 and Gal-1② was measured with human leukemia cells (Jurkat) according to standard protocol. In brief, Jurkat cells in exponential phase were collected by centrifugation at 2000 rpm for 10 min and were suspended at 4 × 105/ml in RPMI 1640 medium containing 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA). One hundred microliter of the suspension was seeded in each well of 96-well plates and serially diluted Gal-1 and Gal-1② were added in quadruplicate, with PBS used as a negative control. After 48 h incubation at 37°C in 5% CO2 in humid air, 20 µl of MTT (0.5 mg/ml) solution was added to each well and cells were cultured for 4 h further. The supernatants in the plates were discarded after centrifugation at 2000 rpm for 10 min, and then 150 µl of DMSO was added. After incubation at room temperature for 10 min, the solubilized reduced MTT was measured at 490 nm using a Bio-Rad plate reader and the optical densities were used to calculate the percentage of death cells with the formula as below: Cell death (%) = (OD control-OD sample) / (OD control) × 100%. Statistical analysis Levels of significance were determined by the Student t test; p values < 0.05 were

9

considered statistically significant. Results Plasmid construction and identification The monomer and concatemer Gal-1 gene of two repeats were amplified by PCR and cloned into pET-22b (+) vector (pET-22b-Gal-1 and pET-22b-Gal-1②) (Fig. 1a and c). After transforming into DH5α E. coli strain, the recombinant plasmids were extracted and identified by enzyme digestion (Fig. 1b and d) and DNA sequencing (data not shown) and results showed that the recombinant genes were correct. Fig.1 Expression and purification of Gal-1 monomer and Gal-1 ② concatemer For recombinant gene expression, the correct plasmids pET-22b-Gal-1 and pET-22bGal-1② were both transformed into BL21 (DE3) strain which can produce T7 RNA polymerase in the presence of IPTG. The recombinant colonies BL21 (DE3) / pET-22b-Gal-1 and BL21 (DE3) / pET-22b-Gal-1② were then cultured and induced with 1 mM IPTG. SDS-PAGE was used to analyze the induced pellets from BL21 (DE3) strain and the results show that a new band about 14.0 kDa or 28.0 kDa was produced in all the soluble bacterial proteins with IPTG induction when compared with non-induced negative controls (Fig. 2a and c). The samples were further identified with Western blot and both of the bands were specifically recognized by Gal-1 monoclonal antibody (Fig. 2b and d). This indicates that both Gal-1 monomer (14.0 kDa) and Gal-1② (28.0 kDa) were successfully expressed in E. coli in soluble forms. 10

Fig.2 Recombinants Gal-1 and Gal-1② were purified by Q- and SP-Sepharose Fast Flow ion-exchange chromatography (Fig. 3 and Fig. 4) and harvested with purity over 95% by HPLC analysis (data not shown). Both of them were obtained with an overall yield about 16% (Table 2).

Fig.3, Fig.4, Table 2 Hemagglutinating activity of Gal-1 monomer and Gal-1 ② concatemer It is well known that one of the characteristics of galectins is their hemagglutination activity, which is attributable to their bivalent carbohydrate-binding property. Given this, erythrocytes agglutination assays were conducted to evaluate purified Gal-1 and Gal-1② and as shown in Fig. 5, recombinant Gal-1 and Gal-1② have activities similar to that of the standard Gal-1 (StGal-1). Both of them can effectively promote agglutination at the dosage of 1.25 µg /well whereas Gal-1② had the highest agglutinating activity. Thus, the agglutination started with as little as 0.16 µg Gal-1②/well. Absence of such activity for PBS used as a negative control confirmed that Gal-1 and Gal-1 ② were indeed responsible for the hemagglutinating activity. Fig.5 Cellular anti-proliferative activity of Gal-1 and Gal-1② To examine the inhibitory effect of Gal-1 and Gal-1② on the proliferation of cells in vitro, we compared the proliferation ratio of Jurkat cells treated with commercially available

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standard Gal-1 (StGal-1), purified recombinants Gal-1 (rGal-1) and Gal-1②. All of them showed remarkable proliferation inhibitory activity towards Jurkat cells when compared with negative control in PBS, whereas Gal-1② had strongest effect at the same wet weight dosages ( p < 0.01) (Fig. 6). If the effect of equimolar molecules were compared, Gal-1② would show even higher such activities. MTT assay results show that Gal-1② at a dosage as low as 200 µg/ml can induce 60% cell death. Fig.6 Discussion It is well accepted that galectin-1 plays an important role in various autoimmune diseases and has been used in clinical trials. But accumulating data has shown that it is only when high dosage is used that Gal-1 can obtain satisfactory results in animal models. If used in humans, the daily dose is about 4 mg/kg. Thus, it is essential to find a way to prepare Gal-1 with higher activity before this molecule can be widely used in clinical trials. Galectin-1 is distributed in the human body as a monomer or homodimer, whereas the latter has stronger activity than the former in maintaining homeostasis of the immune system. Patrick Bättig and his colleagues expressed recombinant wild type and tandem repeats of galectin-1 in 293-EBNA cells [30], and the structurally optimized molecules were found to be much more active in inducing the apoptosis of murine thymocytes and mature T lymphocytes. To sum up, in the present study, we mimicked the strategy of tandem repeat type galectins to construct prototype Gal-1, and used the restriction enzyme recognition site

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BamH I as a short linker in translated protein molecules. The recombinant Gal-1② was efficiently and stably expressed in E. coli at high level (about 30% of all the bacterial proteins) and was conveniently purified by ion-exchange chromatography. Our analysis of the purified Gal-1② shows that it has stronger hemagglutinating activity and can more effectively induce Jurkat cell apoptosis as compared with the standard Gal-1. The specific mechanism for the stronger effect and the pharmacokinetics of gal-1② are still under further investigation. Acknowledgements This project was supported by the Natural Science Fund of China (Project No. 31000406, 81373201) and the grant from the National High Technology Research and Development Program of China (No. 2012AA02A407).

Conflict of Interest The authors declare that there are no conflicts of interest.

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Figure legends Fig.1. Schematic diagram of pET-22b-Gal-1 and pET-22b-Gal-1②. (A and C), The recombinant genes encoding Gal-1 and Gal-1② were cloned into the pET-22b(+) vector and expressed in E. coli BL21(DE3) under the control of T7 promoter. The Gal-1 concatemer was linked by a BamH I enzyme recognition sequence which encodes glycine and asparagine (GN). (B and D), Identification of recombinant plasmids pET-22b-Gal-1 and pET-22b-Gal-1② by restriction enzyme digestion. Lane M, DL2000 DNA marker; lane 1, plasmid pET-22b-Gal-1 (B) and pET-22b-Gal-1② digested with Nde I and Sal I. Fig.2. Expression and identification of Gal-1 and Gal-1②. (A and C), Expression of Gal-1 (A) and Gal-1② (C) in E. coli BL21(DE3) strain. Lane M, molecular weight standards (kDa); lane 1, total bacterial proteins before IPTG induction; lane 2, total bacterial proteins after 1 mM IPTG induction. Arrowheads indicate the target recombinant protein. (B and D), Identification of expressed Gal-1 (B) and Gal-1② (D) by Western blot analysis. Total bacterial proteins of BL21 (DE3) strain without (uninduced) or with (induced) IPTG induction were separated by SDS-PAGE and then transferred onto PVDF membrane and identified by Gal-1 monoclonal antibodies.

Fig.3. Purification of Gal-1 by ion-exchange chromatography. (A and B), The elution profiles of Q-Sepharose (A) and SP-Sepharose (B) Fast Flow chromatography for recombinant Gal-1 purification. Arrowheads indicate the Gal-1 fractions. (C), Analysis of Gal-1 expression and purification by SDS-PAGE. M, molecular weight standards (kDa); lane 1, total bacterial lysate before IPTG induction; lane 2, total bacterial lysate after 1 mM IPTG induction; lane 3, the precipitate of cell lysate; lane 4, supernatant of cell lysate; lane 5, Gal-1 fraction after Q-Sepharose Fast Flow chromatography; lane 6, recombinant Gal-1 after SP-Sepharose Fast Flow chromatography.

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Fig.4. Purification of Gal-1② by ion-exchange chromatography. (A and B), The elution profiles of Q-Sepharose (A) and SP-Sepharose (B) Fast Flow chromatography for recombinant Gal-1 ② purification. Arrowheads indicate the Gal-1② fractions. (C), Analysis of Gal-1② expression and purification by SDS-PAGE. M, molecular weight standards (kDa); lane 1, total bacterial lysate before IPTG induction; lane 2, total bacterial lysate after 1 mM IPTG induction; lane 3, the precipitate of cell lysate; lane 4, supernatant of cell lysate; lane 5, Gal-1② fraction after Q-Sepharose Fast Flow chromatography; lane 6, recombinant Gal-1② after SP-Sepharose Fast Flow chromatography.

Fig.5. Hemagglutinating activity analysis of Gal-1 monomer and Gal-1② concatemer. Erythrocytes were seeded in 96-well plates and treated with twofold serial diluted standard Gal-1 (StGal-1), recombinant Gal-1 (rGal-1) or recombinant Gal-1 concatemer (Gal-1②) for 2 h at room temperature, and each well was examined visually by eyes for complete agglutination. Recombinant protein titer is reported as the reciprocal of the highest dilution that can cause detectable agglutination of erythrocytes. ConA and PBS were used as positive and negative controls, respectively. Data are representative of three experiments.

Fig.6. Gal-1 and Gal-1② inhibit the proliferation of human leukemia cells. Jurkat cells were seeded in 96-well plates and treated with serially diluted StGal-1, rGal-1 and Gal-1② in quadruplicate, PBS was used as a negative control. Cell proliferation was determined by the MTT viability assay. *p