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Contents lists available at ScienceDirect

Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 5 6

Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies

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Zhonglan Hu a,1, Zhenyu Chen a,1, Nongyu Huang a,1, Xiu Teng a, Jun Zhang b, Zhen Wang a, Xiaoqiong Wei a, Ke Qin a, Xiao Liu a, Xueping Wu a, Huan Tang a, Xiaofeng Zhu a, Kaijun Cui c,⇑, Jiong Li a,⇑ a b

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c

State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People’s Republic of China State Key Laboratories of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, People’s Republic of China Cardiology Department, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 8 August 2014 and in revised form 28 October 2014 Available online xxxx Keywords: IL-38 Fusion protein Polyclonal antibodies

a b s t r a c t Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl b-D-1–thio galactoside (IPTG) at 37 °C for 4 h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined. Ó 2014 Published by Elsevier Inc.

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Introduction

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Discovered in silico in 2001, the IL-38 gene is located near genes of other IL-1 family members on human chromosome 2. Specifically, IL-38 is adjacent to the genes encoding IL-1 receptor antago-

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⇑ Corresponding authors at: Department of Cardiovascular Medicine, West China Hospital, Sichuan University, No. 37 Guo Xue Xiang, Chengdu, Sichuan 610041, People’s Republic of China (K. Cui). State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Ke-yuan Road 4, No. 1, Gao-peng Street, Chengdu, Sichuan 610041, People’s Republic of China (J. Li). E-mail addresses: [email protected] (K. Cui), [email protected] (J. Li). 1 Zhonglan Hu, Zhenyu Chen and Nongyu Huang contributed equally to this work. 2 Abbreviations used: IL-1Ra, interleukin-1 receptor antagonist; PBMC, peripheral blood mononuclear cells; LB, Luria–Bertani; PBS, phosphate-buffered saline; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CBB-R250, Coomassie brilliant blue R-250; OD600, optical density at 600 nm; RP-HPLC, reverse phase high-performance liquid chromatography.

nist (IL-1Ra2) and IL-36Ra, and the genomic organization of the IL-38 gene is highly conserved with other IL-1 family members [1–3]. IL-38 is a novel IL-1 family member that shares significant amino acid sequence similarity with IL-1Ra (37%) and IL-36Ra (43%). The natural N terminus of IL-38 is unknown and there is no caspase-1 consensus cleavage site. IL-38 is secreted from cells and lacks a classical signal peptide. Neither N-glycosylation nor O-glycosylation has been detected in this protein. Immunohistochemical analysis showed that IL-38 protein is highly expressed in the basal epithelia of human skin and in proliferating B cells of the tonsil [1]. Semiquantitative PCR analysis showed that IL-38 genes are expressed in fetal skin and at a lower level in spleen [1]. Using multi-tissue first-strand cDNA PCR analysis, IL-38 mRNA was found to be expressed in the heart, placenta, fetal liver, spleen, thymus, and tonsil [4]. The three-dimensional structure of IL-38 is predicted to be similar to that of IL-36Ra. Using a panel of soluble members of the IL-1

http://dx.doi.org/10.1016/j.pep.2014.10.016 1046-5928/Ó 2014 Published by Elsevier Inc.

Please cite this article in press as: Z. Hu et al., Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.016

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receptor family [IL-1R type I, IL-1Rrp2-Fc (IL-36 receptor -Fc), IL-18 Ra chain-Fc, IL-1R accessory protein-Fc] IL-38 was found to bind to IL-1Rrp2-Fc and not to any of the other receptors studied [5–7]. IL38 binds to the IL-36R and has biological effects on immune cells similar to IL-36Ra. Co-stimulation of human peripheral blood mononuclear cells (PBMC) with IL-36c and IL-38, reduced IL-8 production by 42%. By comparison, the effect of IL-36Ra on IL-36-driven IL-8 secretion, was a 73% reduction [5,6]. Genetic association studies indicate that IL-38 might be involved in the pathogenesis of human inflammatory diseases, and allele combinations that include IL-38 polymorphisms are associated with psoriatic arthritis and ankylosing spondylitis [8–10]. Furthermore, IL-36Ra and IL-38 reduce candida-induced Th17 responses [5]. Recent studies have shown that aberrant IL-36Ra structure and function leads to unregulated secretion of inflammatory cytokines and generalized pustular psoriasis [11,12]. IL-38 may play an important role in the pathogenesis of psoriasis, and therefore, exploring the role of IL38 in this disorder may lead to the development of novel treatments. Hence, data suggest that IL-38 is a negative regulator involved in human inflammation and autoimmunity. Further work is required to validate this claim and to dissect the mechanism of IL-38 action. Herein, we describe the construction of a pEHISTEV-IL-38 vector and optimization of the conditions required for its expression, and purification of IL-38 in Escherichia coli. Beyond that, we develop specific antibodies for use in future research.

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Materials and methods

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Bacterial strains, plasmids, and growth media

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E. coli strain DH5a was used for gene cloning and E. coli BL21 (DE3) was used to express the fusion protein. The cells harboring expression plasmids were cultured in Luria–Bertani (LB) medium supplemented with kanamycin (100 lg/mL).

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Construction of fusion expression plasmid pEHisTEV-IL-38

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We optimized the coding region of IL-38 DNA (NM_032556.5) to enable expression in an E. coli system. The sequence was synthesized in GenScript (Nan Jing, China), and was cloned into the pEHISTEV vector which we optimized using PCR primers (50 -GACACCATGGGTTCCCTGCCGATGGCT-30 and 50 AGTGGTGGTGGTGGTGGTGC 30 ). PCR was carried out using the high-fidelity Pyrobest DNA polymerase (TaKaRa). The PCR programme consisted of a DNA denaturation step at 94 °C for 5 min followed by 25 cycles at 94 °C for 30 s, 58 °C for 20 s, and 72 °C for 30 s and a final elongation step at 72 °C for 10 min. The products, which included the sequence encoding a TEV protease site adjacent to the IL-38 coding sequence, were analyzed on a 1% agarose gel stained with Goldview, and electrophoresis was performed in 1  TAE buffer, at 120 V for 25 min. Following electrophoresis, the DNA band corresponding to the correct size of the desired gene was excised from the gel. Next, PCR products were extracted from the gel and purified using quick PCR purification kit (Sigma), followed by digestion with NcoI and XhoI restriction enzyme (TaKaRa). The digested PCR products were ligated into the pEHISTEV vector at NcoI/XhoI sites using T4 DNA ligase (TaKaRa) at 16 °C overnight, and the ligated constructs were transformed into E. coli DH5a cells cultured under kanamycin. We selected a kanamycin-resistant clone, which was then subcultured in 10 mL LB liquid medium containing kanamycin (100 lg/mL). Plasmid DNA was extracted using a Plasmid mini kit (Omega). The sequence of the IL-38 coding region was confirmed by DNA sequencing (Life Technology Inc, Shanghai, China).

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Optimization of expression conditions and solubility testing

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E. coli strain BL21 (DE3) was used to express the fusion protein. First, the pEHisTEV-IL-38 expression vectors were transformed into E. coli BL21 (DE3) cells, a positive colony was selected from an LB agar plate supplemented with kanamycin (100 lg/mL), and small-scale expression was used to optimize the experimental conditions. Transformed E. coli were cultured in 10 mL LB liquid medium containing kanamycin (100 lg/mL) overnight at 37 °C, with shaking at 220 rpm/min. The following day, 1 mL bacterial solution was inoculated into 19 mL fresh LB liquid medium containing kanamycin (100 lg/mL), and then subcultured by splitting into three separate vessels. Pre-cultures were grown until an absorbance at 600 nm of 0.6–0.8 was reached, then, 0.1 mM IPTG was added to each subculture and expression of fusion proteins was induced at 16 °C, 25 °C, and 37 °C for 4 h. Next, cells were stimulated with 0.5 mM IPTG at 37 °C for 4 h in an attempt to increase the expression level of the fusion protein. Subcultures were then collected by centrifugation at 5000 rpm for 15 min at 4 °C, and pellets were resuspended in phosphate-buffered saline (PBS). Cells were sonicated on ice with an ultrasonic disintegrator (Ningbo XinYi Co, Ltd, China), following which cell suspensions were centrifuged at 15,000g for 3 min at 4 °C. Clear supernatant (soluble fraction), and pellet (insoluble fraction) were collected and analyzed on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) followed by Coomassie brilliant blue R-250 (CBB-R250) staining. The control cultures were analyzed in parallel [13–15].

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Expression and purification of the fusion protein

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A positive single colony was inoculated into 40 mL of LB media containing kanamycin (100 lg/mL) and grown overnight at 37 °C. The overnight cultures were diluted 1:100 into 1 L LB medium with kanamycin (100 lg/mL), and cells were grown at 37 °C with shaking until the optical density at 600 nm (OD600) reached 0.6–0.8, after which protein expression was induced with 0.1 mM IPTG for 4 h. The expression level was analyzed by 15% SDS–PAGE. Cells were harvested by centrifugation (5000 rpm, 15 min). The cell pellets from 1 L of culture were resuspended in 30 mL PBS and lysed by high-pressure homogenization. The cell lysates were cleared by centrifugation at 15,000 rpm for 30 min at 4 °C, and the supernatant was filtered through a 0.45-lm filter (Millipore). The filtered supernatant was loaded onto a 10 mL Ni Sepharose™ fast flow resin column (GE Healthcare) run by AKTA explorer (GE Healthcare), and was pre-equilibrated with PBS at a flow rate of 3 mL/min until the absorbance at 280 nm stabilized at baseline. The protein bound to the column was eluted with different concentrations of elution buffer (PBS containing different concentrations of imidazole, pH 7.8). SDS–PAGE was used to analyze the purity of the eluted fusion protein following CBB R-250 staining. Then, the eluted protein was dialyzed against PBS using Sephadex G-25M (GE Healthcare) [13–15]. Final concentrations of purified fusion proteins were determined with a NanoDrop-8000 Spectrophotometer (Thermo Scientific).

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Purification of IL-38 protein

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TEV protease purified by our lab was added to the fusion protein, and then incubated at 4 °C and at room temperature to optimize enzymatic digestion. After overnight incubation, SDS–PAGE was used to analyze the digestion efficiency, undigested protein was analyzed in parallel. The protein treated with TEV protease was purified on a Ni Sepharose™ fast flow affinity column and collected pooled fractions of peaks. Next, SDS–PAGE was used to analyze the purity of

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Fig. 1. Construct of the recombinant plasmid pEHISTEV-IL-38. (A) Schematic diagram of the IL-38 fusion protein construct. The expression construct contains a N-terminal histidine-tag, a TEV protease site, and the IL-38 gene sequence. (B) Sequence alignment. The result from DNA sequencing were consistent with the optimized DNA sequence.

Fig. 2. SDS–PAGE analysis was performed to confirm expression of the fusion protein pETHisTEV-IL-38 in E. coli BL21 (DE3) under different conditions. Lane M, protein markers. Lane 1, non-induced. Lane 2, E. coli BL21 transformants incubated with 0.1 mM IPTG for 4 h at 16 °C. Lane 3, induction with 0.1 mM IPTG at 16 °C overnight. Lane 4, induction with 0.1 mM IPTG for 4 h at 25 °C. Lane 5, induction with 0.1 mM IPTG for 4 h at 37 °C. Lane 6, induction with 0.5 mM IPTG for 4 h at 37 °C.

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recombinant IL-38. Furthermore, we used molecular-exclusion chromatography on a Superdex 75 column (GE Healthcare) for further purification. After pre-equilibration of the column with five bed volumes of equilibration buffer (20 mM Hepes-Na, 0.1 M NaCl pH7.2), sample was loaded onto the column. Columns were then washed with wash buffer (20 mM Hepes-Na, 0.1 M NaCl pH 7.2, flow rate 0.3 mL/min). The void volume and eluted fractions from the column were monitored at 280 nm. Pooled fractions of peaks were collected and subjected to SDS–PAGE analysis [11–13]. Reverse phase high-performance liquid chromatography (RP-HPLC) (Waters) was then used to determine its purity.

Binding array of IL-38 and IL-36R

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Next, we measured the binding ability of IL-38 and IL-36R using a functional ELISA [5]. The wells of a 96-well ELISA plate were coated with IL-38 protein diluted with carbonate coating buffer (0.05 mol/L, pH 9.6) at 1 lg/mL and incubated at 4 °C overnight. After washing, samples were blocked with 1% BSA in PBS at 37 °C for 2 h. Serial dilutions of recombinant human IL-36R/Fc Chimera (R&D systems) were prepared using 1% BSA in PBS. Samples were added to the ELISA plate, along with the control group that received the same amount of PBS, and was incubated at 37 °C for 2 h. Bound protein was detected by antibodies conjugated to HRP specific to human IgG Fc (Sino Biological Inc.), followed by TMB-S program (Boster, Wuhan, China). Absorbance of samples was read at 450 nm in a UV–visible plate reader (Thermo Scientific) [16,17].

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Production and purification of polyclonal antibodies against IL-38

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Purified protein was used to raise antibodies in a New Zealand white rabbit. The rabbit was inoculated with 200 lg of IL-38 protein in Freund’s complete adjuvant (Sigma). Before the primary immunization, a blood sample was collected from the ear vein, and the serum was used for titer determination of antibodies. Two weeks later, the rabbit received three boosters of 200 lg protein in incomplete Freund’s adjuvant (Sigma) at two-week intervals [18]. Serum was obtained one week before the final immunization and antibody titer was determined by ELISA using the pre-immu-

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Fig. 5. SDS–PAGE analysis following optimization of TEV protease digestion and protein purification. Lane M, protein markers. Lane 1, the desalted pre-digested recombinant protein. Lane 2, protein cleaved with TEV protease overnight at 4 °C or at room temperature (Lane 3).

Fig. 3. SDS–PAGE analysis of the solubility of the fusion protein induced under different conditions. The protein was mainly distributed in the supernatant. (A) Detection of the solubility of recombinant IL-38 induced with 0.1 mM IPTG for 4 h or overnight at 16 °C. Lane M, protein markers. Lane 1 and 4, clarified lysate after centrifugation. Lane 2 and 5, pellet after centrifugation. Lane 3 and 6, total cell lysate. (B) Determination of the solubility of IL-38 fusion protein induced with 0.1 mM IPTG for 4 h at 25 °C, and 0.1 mM IPTG and 0.5 mM IPTG at 37 °C for 4 h. Lane M, protein markers. Lane 1, 4 and 7, total cell lysates. Lane 2, 5 and 8, supernatant after centrifugation. Lanes 3, 6 and 9, pellet after centrifugation. Lanes 1–3, the sample diluted three times. Lanes 4–9, the sample was diluted four times. Fig. 6. SDS–PAGE analysis of purified IL-38. (A) Protein digested with TEV protease was purified by affinity column. Lane M, protein markers. Lane 1, protein treated with the reducing buffer; and non-reducing buffer (lane 2). (B) Protein was further purified by molecular-exclusion chromatography. Lane M, protein markers. Lane 1, IL-38 analyzed with reducing loading buffer, and non-reducing loading buffer (lane 2).

Fig. 4. SDS–PAGE analysis of purified fusion protein. Lane M, protein markers. Lane 1, uninduced cells. Lane 2, induced cells after 4 h with 0.1 mM IPTG. Lane 3, total cell lysates. Lane 4, supernatant from cell lysates. Lane 5, precipitate after centrifugation. Lane 6, flow-through of the affinity column. Lane 7, the eluted protein with elution buffer (PBS containing 30 mM imidazole). Lane 8, the eluted protein with elution buffer (PBS containing with 100 mM imidazole). Lane 9, the eluted protein with elution buffer (PBS containing 250 mM imidazole). Lane 10, the eluted protein with elution buffer (PBS containing 500 mM imidazole). 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239

nized rabbit serum as a negative control. Ten days after the final immunization, blood was taken from the rabbit heart, and the final Q4 titer of antiserum was determined by ELISA [19,20]. Next, the antibody was purified using rProtein A Sepharose™ (GE Healthcare) according to its instruction. The purified antibody dialyzed against PBS and protein concentration was measured using the BCA protein array kit (Pierce). Samples were packaged and stored at 80 °C until further use. Then, the specificity of polyclonal antibodies was tested by Western blotting. We synthesized the nucleotide sequence for complete coding region of IL-38 (NM_032556.5) for expression in a eukaryotic expression system and cloned into pcDNA3.1 vector in GenScript (Nan Jing, China). the 293A cells were transiently transfected with the pcDNA3.1-IL-38 plasmid or mock-transfected with pcDNA3.1. 24 h later, total protein was extracted by RIPA lysis buffer and analyzed by Western blotting.

Results

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Generation of the recombinant pEHisTEV-IL-38 expression vector

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The IL-38 gene was amplified by PCR and a sequence coding for a TEV protease site was subcloned up stream of the IL-38 gene (Fig. 1A). The PCR products were subjected to analysis by 1% agarose gel electrophoresis, sequentially double digested with NcoI/XhoI restriction enzymes, and subcloned into the pEHisTEV vector. The recombinant vector was transformed into E. coli DH5a cells and double digestion was verified. The successfully constructed pEHisTEV-IL-38 was verified by DNA sequencing (Fig. 1B).

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Optimization of conditions for protein expression

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To express IL-38 in E. coli, the pEHISTEV-IL-38 expression vector was transformed into E. coli BL21 (DE3) cells. Small-scale optimization studies were carried out to identify optimal culture conditions and were subjected to 0.1 mM IPTG treatment at 16 °C, 25 °C, and 37 °C, for 4 h to identify the expression capacity by SDS–PAGE analysis (Fig. 2). As shown in Fig. 2, high expression of fusion protein with the expected molecular weight was observed in BL21 cells compared with the negative controls, when incubated at 37 °C for 4 h. However, the amount of protein produced was very low when incubated at 16 °C for 4 h. The expression level at 16 °C overnight gave a modest increase, but this value remained lower than that induced at 37 °C, and the expression level at 25 °C was moderate. Next, we attempted to increase expression of the fusion protein by increasing the concentration of IPTG. As shown in Fig. 2 (lane 5 and 6), little difference was observed

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Fig. 7. RP-HPLC analysis showing that the purity of IL-38 is greater than 95%.

Table 1 Partial purification of IL-38 and its yield. Step

Volume (mL)

Pellet weight (g)

Total protein (mg)

Fusion IL-38 (mg)

IL-38 (mg)

Step yield (%)

Overall yield (%)

Purity (%)c

Bacterial culture Crude lysate Nickel affinity chromatographya TEV treatment Further purificationb (pooled peak) Molecular-exclusion chromatography (pooled peak)

1000.0 30.0 38.0 38.0 27.0 16.0

2.9 – – – – –

– 256.0 144.0 118.2 43.0 14.9

– 143.4 118.2 – – –

– – – 78.0 37.4 14.6

– – 82.4 – 47.9 39.0

– 100.0 82.4 – 26.1 10.2

– 56.0 82.1 66.0 87.0 98.0

a The supernatant flow through the Ni Sepharose™ and the eluted protein was dialyzed against PBS. Final concentrations of fusion proteins were determined with a NanoDrop-8000 Spectrophotometer. b The fusion protein treated with TEV protease was purified again using affinity chromatography. c The purity of protein in SDS–PAGE was determined by the software of Band scan with Coomassie brilliant blue.

Fig. 8. Binding curves of IL-36R to immobilized IL-38 (n = 3). The data are expressed as mean ± SD.

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between 0.1 mM and 0.5 mM IPTG treatment at 37 °C for 4 h. Furthermore, we analyzed the solubility of the fusion protein by SDS– PAGE. Both the cell supernatant and cell pellet after sonication were analyzed, and the protein was found to be mainly distributed in the supernatant (Fig. 3). SDS–PAGE analysis showed that recombinant IL-38 had an expected molecular mass of 22 kDa. Therefore, we determined that the optimum temperature, IPTG concentration, and induction time were 37 °C, 0.1 mM, and 4 h, respectively, for scale-up production to a 1 L culture.

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Expression and purification of IL-38protein

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A total of 120 mL filtered supernatant was loaded onto a Ni affinity column pre-equilibrated with PBS. The protein bound to the column was eluted with different concentrations of elution buffer (PBS including 30 mM, 100 mM, 250 mM imidazole and 500 mM imidazole, pH 7.8). The purity of the eluted fusion protein

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was analyzed by SDS–PAGE following CBB R-250 staining. As shown in Fig. 4, the fusion protein IL-38 with a histidine tag was mainly eluted with wash buffer (PBS containing with 250 mM imidazole) (Fig. 4, lane 4). A small fraction of eluted fusion protein was dialyzed against PBS and completely cleaved after incubation with TEV protease over night at 4 °C and at room temperature. SDS–PAGE analysis of digestion efficiency revealed that almost all proteins were cleaved adequately (Fig. 5). Based on the above result, the remainder of the protein was digested at 4 °C over night. Fusion protein treated with TEV protease was purified using a Ni Sepharose™ fast flow affinity column. Next, SDS–PAGE was used to analyze the purity of IL-38 with reducing and non-reducing loading buffer (Fig. 6A). IL-38 had an expected molecular mass of 17 kDa. In addition, we used molecular-exclusion chromatography for further purification (Fig. 6B). RP-HPLC was used to analyze purity, which was determined to be greater than 95% (Fig. 7 and Table 1).

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The binding array of IL-38 and IL-36R

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We used a functional ELISA to measure the binding ability of IL-38 and IL-36R [5]. In this assay, the IL-38 purified proteins were immobilized on an ELISA plate, and increasing concentrations of the extracellular domain of an IL-36R/Fc chimera were added. After incubation and washing, HRP-conjugated antibodies specific to human IgG Fc were used to detect bound IL-36R using TMB chromogenic solution. As shown in Fig. 8, the absorbance at 450 nm increased with the concentration of IL-36R, which resulted in a plateau at 16 lg/mL.

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Production and purification of polyclonal antibodies against IL-38

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Purified recombinant proteins were used to generate antibodies in a New Zealand white rabbit as previously described [19,20].

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Fig. 9. Titer determination of polyclonal antibodies by ELISA and specificity testing by Western blotting. The data are expressed as mean ± SD. (A) Absorbance values at 450 nm of rabbit serum taken after the final immunization, and dilution at different ratios. (B) The ratio of anti-serum absorbance values at 450 nm compared with negative serum. (C) Specificity determination by Western blotting. Lane 1, Protein from 293A cells transfected with pcDNA3.1-IL-38 plasmid and mock-transfected with pcDNA3.1 (Lane2).

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Prior to the first immunization, blood samples were taken from the ear vein. Ten days after the last immunization, blood was taken from the rabbit heart, and the final titer of antiserum was detected by ELISA. As shown in Fig. 9A and B, the dilution ratio of serum ranged from1:1000 to 1:128,000, the absorbance value ratio of antiserum to negative serum is 2.12 when the ratio is 1:64,000. Here, we determined when the absorbance value at 450 nm of antiserum to negative serum is greater than 2.1, the maximum dilution ratio is its titer. Therefore, the maximum titer of antiserum is approximately 64,000. Next, we determined the specificity of the generated polyclonal antibody by Western blotting using the protein from 293A cells transfected with the pcDNA3.1-IL-38 plasmid or mock-transfected with pcDNA3.1 lacking IL-38. We found that the polyclonal antibody combined specifically with the former but not mock-plasmid (Fig. 9C).

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Discussion

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Production of soluble recombinant proteins is vital for analysis of their structure, function, and therapeutic application. The IL-38 protein lacks any N-glycosylation consensus sites. Neither Nglycosylation nor O-glycosylation on the recombinant IL-38 protein expressed in CHO cells was detected by peptide N-glycosidase F and O-glycosidase deglycosylation analysis [1]. Furthermore, we predicted IL-38 tertiary structure according to homology modeling using the Molecular Operating Environment (Cloudscientific, Shanghai, China), and found that its tertiary structure is very similar to IL-36Ra (PDB: 1MD6), IL-38 lacked any disulfide bonds in its predicted structure. We used an E. coli expression system to express and purify IL-38 protein. This is a fast and inexpensive way to test a wide variety of possible strategies in E. coli, and one can complete a fairly comprehensive analysis within a relatively short period of time. BL21 (DE3) is an appropriate E. coli strain for use in high-level protein production, it has the advantage of being deficient in both lon and ompT proteases and is compatible with the T7 lacO promoter system33 [11]. In this study, we selected BL21 (DE3) as the host

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bacterial strain in which the recombinant protein was overexpressed after induction with IPTG (Fig. 2). The expression level and solubility of recombinant proteins are major concerns in protein expression. Small-scale test expressions are widely used as a predictive tool to determine the concentration of IPTG and induction time that are most suitable for large-scale growth. Here, we successfully constructed the vector pEHisTEVIL-38, which was transformed into BL21 (DE3) in order to export fusion protein. Next, we treated BL21 (DE3) cells with 0.1 mM IPTG for 4 h respectively, at 16 °C, 25 °C, and 37 °C. Almost no protein was expressed following incubation with 0.1 mM IPTG for 4 h at 16 °C, but high protein expression was observed following incubation at 37 °C (Figs. 2 and 3). Next, the induction time was extended and samples were incubated. However, under these conditions expression of the fusion protein was lower than that obtained at 37 °C. In addition, we attempted to increase expression by increasing the concentration of IPTG at 37 °C, but similarly, there was not much difference between these conditions. Therefore, the conditions used to induce protein expression were 37 °C, 0.1 mM IPTG for 4 h for scale-up productions. IL-38 binds to the IL-36 receptor and has biological effects on immune cells similar to IL-36Ra [5]. Here, we tested the binding ability between IL-38 and IL-36R, our result is consistent with previous report. In conclusion, in this study we obtained high purity protein after size exclusion chromatography. Furthermore, we isolated a highly specific polyclonal antibody to enable further investigation into the mechanism of IL-38 action and structure.

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Acknowledgments

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This work was supported by China National Science and Technology Programs of Prophylaxis and Therapy of Significant Infec- Q5 tious Diseases (No. 2012ZX10002006-003-001), China National Science and Technology Programs of Significant New Drugs to Create (No. 2013ZX09301304-003), National Natural Science Q6 Foundation of China (No. 31271483).

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8 November 2014 Z. Hu et al. / Protein Expression and Purification xxx (2014) xxx–xxx

References

383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409

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[1] Haishan Lin, Alice S. Ho, Dana Haley-Vicente, Jun Zhang, et al., Cloning and characterization of IL-1HY2, a novel interleukin-1family member, J. Biol. Chem. 276 (23) (2001 Jun 8) 20597–20602. [2] C. Dinarello, W. Arend, J. Sims, D. Smith, H. Blumberg, et al., IL-1 family nomenclature, Nat. Immunol. 11 (11) (2010 Nov) 973. [3] J.E. Sims, M.J. Nicklin, J.F. Bazan, J.L. Barton, et al., A new nomenclature for IL-1family genes, Trends Immunol. 22 (10) (2001 Oct) 536–537. [4] J.T. Bensen, P.A. Dawson, J.C. Mychaleckyj, D.W. Bowden, Identification of a novel human cytokine gene in the interleukin gene cluster on chromosome 2q12-14, J. Interferon Cytokine Res. 21 (11) (2001 Nov) 899–904. [5] F.L. van de Veerdonk, A.K. Stoeckman, G. Wu, A.N. Boeckermann, et al., IL-38 binds to the IL-36 receptor and has biological effects on immune cells similar to IL-36 receptor antagonist, Proc. Natl. Acad. Sci. U.S.A. 109 (8) (2012 Feb 21) 3001–3005. [6] C. Garlanda, C.A. Dinarello, A. Mantovani, The interleukin-1 family: back to the future, Immunity 39 (6) (2013 Dec 12) 1003–1018. [7] A. Dehghan, J. Dupuis, M. Barbalic, J.C. Bis, G. Eiriksdottir, et al., Meta-analysis of genome-wide association studies in >80000 subjects identifies multiple loci for C-reactive protein levels, Circulation 123 (7) (2011 Feb 22) 731–738. [8] W.I. Lea, Y.H. Lee, The associations between interleukin-1 polymorphisms and susceptibility to ankylosing spondylitis: a meta-analysis, Joint Bone Spine 79 (4) (2012 Jul) 370–374. [9] W.P. Maksymowych, P. Rahman, J.P. Reeve, D.D. Gladman, L. Peddle, R.D. Inman, Association of the IL1 gene cluster with susceptibility to ankylosing spondylitis: an analysis of three Canadian populations, Arthritis Rheum. 54 (3) (2006 Mar) 974–985.

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[10] S. Marrakchi, P. Guigue, B.R. Renshaw, A. Puel, et al., Interleukin-36-receptor antagonist deficiency and generalized pustular psoriasis, N. Engl. J. Med. 365 (7) (2011 Aug 18) 620–628. [11] B.M. Hallberg, J. Bray, O. Gileadi, S. Knapp, U. Oppermann, et al., Protein production and purification, Nat. Methods 5 (2) (2008 Feb) 135–146. [12] J. Arnau, C. Lauritzen, Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags, Nat. Protoc. 1 (5) (2006) 2326–2333. [13] J.M. Fleckenstein, K. Roy, Purification of recombinant high molecular weight two-partner secretion proteins from Escherichia coli, Nat. Protoc. 4 (7) (2009) 1083–1092. [14] G. Saletti, N. Çuburu, J.S. Yang, A. Dey, C. Czerkinsky, Enzyme-linked immunospot assays for direct ex vivo measurement of vaccine-induced human humoral immune responses in blood, Nat. Protoc. 8 (6) (2013 Jun) 1073–1087. [15] S.S. Pierangeli, E.N. Harris, A protocol for determination of anticardiolipin antibodies by ELISA, Nat. Protoc. 3 (5) (2008) 840–848. [16] H. Kothari, P. Kumar, N. Singh, Prokaryotic expression, purification, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate, Protein Expr. Purif. 45 (1) (2006 Jan) 15– 21. [17] C. Wu, Y. Wang, M. Zou, Y. Shan, G. Yao, et al., Prokaryotic expression, purification, and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyltransferase 14, Protein Expr. Purif. 56 (1) (2007 Nov) 1–7. [18] J. Yang, S.Y. Guo, F.Y. Pan, H.X. Geng, et al., Prokaryotic expression and polyclonal antibody preparation of a novel Rab-like protein mRabL5, Protein Expr. Purif. 53 (1) (2007 May) 1–8.

Please cite this article in press as: Z. Hu et al., Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies, Protein Expr. Purif. (2014), http://dx.doi.org/10.1016/j.pep.2014.10.016

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Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.

Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member o...
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