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IL7(2009)Expression, purification, and functional characterization of recombinant human interleukin-


Protein Expression and Puri?cation 63 (2009) 1–4

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Protein Expression and Puri?cation
journal homepage: www.elsevier.com/locate/yprep

Expression, puri?cation, and functional characterization of recombinant human interleukin-7
Yong Luo a,b, Xiangping Kong c, Aimin Xu a,d, Shouguang Jin e, Donghai Wu a,b,*
a

Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Science Park, Guangzhou 510663, China Department of Life Sciences, University of Science and Technology of China, China c Key Laboratory of Liver Diseases Research of 458th Hospital, China d Department of Medicine, The University of Hong Kong, Hong Kong, China e Department of Molecular Genetics and Microbiology, University of Florida, USA
b

a r t i c l e

i n f o

a b s t r a c t
Human interleukin-7 (IL-7) is a member of the interleukin family. Numerous studies have demonstrated IL-7’s effect on B- and T-cell development as well as its potential in various clinical applications. Previously, a study reported that IL-7 could be puri?ed from inclusion bodies using a prokaryotic system, however, the required refolding step limits the recovery rate. This study was designed to produce a bioactive recombinant human IL-7 (rhIL-7) in a eukaryotic expression system in order to obtain higher yields of the protein with simpler puri?cation steps. We cloned human IL-7 cDNA and successfully expressed active recombinant protein in yeast using the Pichia pastoris expression system. A simple puri?cation strategy was established to purify the rhIL-7 from the fermentation supernatant, yielding 35 mg/L at 95% purity by the use of a common SP Sepharose FF cation-exchange chromatography. Functional analysis of the puri?ed rhIL-7 by the pre-B cell MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) proliferation assay demonstrated a speci?c activity comparable to commercial sources. These results suggest that puri?cation of rhIL-7 from yeast provides a sound strategy for large-scale production of the rhIL7 for clinical applications as well as basic researches. ? 2008 Elsevier Inc. All rights reserved.

Article history: Received 6 March 2008 and in revised form 19 June 2008 Available online 6 September 2008 Keywords: Pichia pastoris Human interleukin-7 Recombinant protein Puri?cation SP Sepharose Cell proliferation

Interleukin-7 (IL-7)1 is a member of the interleukin family which regulates the development and survival of lymphoid and myeloid cells. It was ?rst discovered in 1988 as a factor that promotes the growth of murine B cell precursors in a bone marrow culture system [1,2]. Later studies showed that injection of mice with IL-7 resulted in elevated numbers of T and B lymphocytes [1,3]. Its importance in lymphoid development was con?rmed by the lymphopenic phenotypes of IL-7 and IL-7 receptor knockout mice as well as humans with defects in the IL-7 receptor [4–6]. Recombinant human IL-7 (rhIL-7) is a 152 amino acids long single-chain polypeptide with a molecular weight of 17.4 kDa. The N-terminal region of the protein contains a signal sequence for secretion. Although the crystal structure of IL-7 remains to be determined, its three-dimensional structure has been predicted based on homology to known structures of other family members such as IL-2 and IL-4 [7,8]. The predicted model contains a compact
* Corresponding author. Address: Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Science Park, Guangzhou 510663, China. Fax: +86 20 32290269. E-mail address: wu_donghai@gibh.ac.cn (Donghai Wu). 1 Abbreviations used: IL-7, interleukin-7; rhIL-7, recombinant human IL-7; YNB, yeast nitrogen base; YPD, yeast extract peptone dextrose; FBS, fetal bovine serum; DMSO, dimethylsulfoxide. 1046-5928/$ - see front matter ? 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2008.08.009

hydrophobic core and has a topology common to other four-helix bundle proteins. Although the IL-7 receptor has a similar structure to other receptors from members of the interleukin family, IL-7 plays a unique role in the thymus and homeostasis of na?ve peripheral T cells [9–12]. The biological functions of IL-7 as a key ‘‘lymphotrophin” in JAK-STAT pathways, PI3 kinase pathway, and Src kinase pathway have been reviewed by Durum et al. [13]. More recently, IL-7 has also been shown to play a vital role in energy metabolism of T cells and regulating glucose uptake in T lymphocytes through Glut1 via activation of STAT5 and Akt [14]. Clinical studies of IL-7 for treatment of various diseases, including immunode?ciency disorders and malignancies are underway, with promising results suggesting that IL-7 may facilitate additional therapeutic endeavors including bone marrow/organ transplantation and cancer immunotherapy [15–18]. Currently, human IL-7 is mainly produced from an Escherichia coli expression system which involves inclusion bodies that requiring a refolding step [19]. In this study, we established a more ef?cient method to produce large quantities of rhIL-7 with high bioactivity using a eukaryotic expression system of Pichia pastoris. This will facilitate future studies of its structure–function relationship as well as clinical applications.

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Y. Luo et al. / Protein Expression and Puri?cation 63 (2009) 1–4

Materials and methods Strains and reagents Pichia pastoris strain X-33, expression vector pPICZaB, Yeast nitrogen base (YNB), D-biotin, and MTT assay kits were purchased from Invitrogen (USA). Plasmid extract kit, restriction enzymes, dNTP, pfu DNA polymerase, and T4 DNA ligase were obtained from Takara (Guangzhou, China). SP Sepharose and ECL Western blot detection kit were obtained from Amersham Biosciences (USA). Anti-human IL-7 monoclonal antibody was purchased from R&D (USA) and 2E8 cell line was from ATCC (USA). Other reagents were obtained from standard commercial sources and were of Reagent grade. Plasmid constructs Human IL-7 cDNA (459 bp) was ampli?ed by PCR from a plasmid encoding full-length human IL-7 (cat. MHS1010-9205059, Open Biosystem, USA). To express the native N terminus of IL7, a XhoI site was introduced to allow in-frame cloning into afactor secretion signal of pPICZaB and a nucleotide sequence encoding the KEX2 cleavage site was placed upstream of the IL-7. Forward and reverse primers used were 50 -GGCTC GAG AAA AGA GAT TGT GAT ATT GAA GGT AAA-30 and 50 -AGTCT AGA TCA GTG TTC TTT AGT GCC CAT CAA AT-30 , respectively. The ampli?ed gene was con?rmed by sequencing with ABI PRISM 310 sequencer. The PCR products were digested with XhoI and XbaI and inserted between the XhoI and XbaI sites of pPICZaB where the IL-7 is under the control of alcohol oxidase1 promoter on the vector. Transformation of P. pastoris and selection of transformants Pichia pastoris X-33 was transformed with a linearized expression vector by digesting with SacI. Transformation was performed using the lithium chloride method as described in the P. pastoris expression manual (Invitrogen, USA). Transformants were plated on a yeast extract peptone dextrose (YPD) plate containing 100 lg/ml of Zeocin. Zeocin-resistant colonies were replated on YPD plates containing 500 lg/ml of Zeocin. After incubation at 30 °C for 48 h, large colonies were selected for further analysis. Small-scale fermentation and time course expression study Selected clones were grown in 25 ml of BMGY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen broth, 4 ? 10?5% biotin, 1% glycerol), until the OD600 measured >2.0 (between 2.0 and 6.0). The cultures were centrifuged for 15 min at 2500g and pellets were collected. For the induction phase, the collected cell pellets were inoculated into 250 ml of BMMY media (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen broth, 4 ? 10?5% biotin, 1% methanol) in Baf?ed ?asks and grown for 4 days, as described by the manufacturer (Invitrogen). Each day, 5 ml of 100% methanol was added per liter of culture (?nal 0.5%) to induce the protein expression. Expression of the protein was monitored by SDS–PAGE (12% gel) separation of the culture media followed by Coomassie blue stain. Western immunoblot was also performed to con?rm the identity of the secreted protein using mouse anti-hIL-7 monoclonal antibody. Levels of rhIL-7 in the culture media were monitored by subjecting 10 ll of the supernatant to the Western blot analysis in time course studies.

Large-scale fermentations and puri?cation procedure Large-scale fermentations were carried out in a 30 L working volume reactor (Bio?o 4500, New Brunswick Scienti?c). Sixteen liters of basal salts medium (4.25 ml/L orthophosphoric acid, 9.4 mM MgSO4, 1 mM CaSO2, 16.4 mM K2SO4, 11.4 mM KOH, and 50 ml/L glycerol) were sterilized in the reactor. Ammonium was used as a pH control reagent, while at the same time serves as nitrogen source. PTM1 trace salts (2 ml/L; 24 mM CuSO4, 0.53 mM NaI, 19.87 mM MnSO4, and 0.83 mM Na2MoO4, 0.32 mM boric acid, 2.1 mM CoCl2, 0.15 mM ZnCl2, 0.23 M FeSO4, and 0.82 mM biotin) were added after sterilization. P. pastoris harboring the rhIL-7 expression vector (pPICZaB-hIL-7) was grown in one liter of BMGY overnight and inoculated into the medium in fermentor. Oxygen was maintained at 35% and pH was at 5.0 during the growth phase. During fermentation, a glycerol-fed-batch phase was carried out in which biomass was bulked up to the desired cell density using 50% glycerol feed containing 12 ml/L PTM1. Induction was carried out by feeding methanol (containing 12 ml/L PTM1) at a gradient feed rate into the fermentor and pH was maintained at 5.0. Induction was maintained for approximately 56 h before harvesting. The culture medium was centrifuged for 15 min at 5000g and the supernatant was concentrated by ultra?ltration against buffer A (20 mM sodium phosphate buffer, pH 5.0). Then, the samples (3.5 L) were loaded onto SP Sepharose FF cation-exchange resin (25 ml, from Amersham Biosciences, and the Column size is 2.5 ? 30 cm (Bio-Rad)), washed and eluted with linear gradient of 0–2.0 M NaCl in PBS. The eluted protein was analyzed by SDS–PAGE. Western blot Protein samples were separated on 15% SDS–PAGE gels and electroblotted onto PVDF membranes. The membrane was blocked with 5% milk in 10 M Tris–HCl with 150 mM NaCl (pH8.0) and 0.1% Tween 20 (TBST) for 2 h at room temperature. The membrane was then incubated for 2 h at room temperature with mouse anti-hIL-7antibody (diluted 1:2000 in TBST with 5% milk). After three washes, the membrane was incubated for 1 h with peroxidase-conjugated secondary antibody (goat anti-mouse IgG, diluted 1:5000 in TBST). The membrane was washed three times with TBST and the speci?c protein bands were visualized by ECL detection kit (Amersham Biosciences). MTT proliferation assay The biological activity of rhIL-7 was determined by its growthpromoting action on 2E8 cell line. The 2E8 cell line was maintained in IMDM medium (GIbco, Grand Island, NY) containing 5% fetal bovine serum (FBS, Hyclone). To initiate cellular proliferation, different concentrations (1 pg/ml–100 ng/ml) of rhIL-7 were incubated with 2 ? 105 per well 2E8 cells in 96-well plates. After incubation for 48 h, cell proliferation was detected by the MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay as described [20]. Brie?y, 10 ll of MTT solution (5 mg/ml MTT in IMDM) was added into each well of 96-well plates. The plates were incubate at 37 °C for 4 h and then centrifuged at 8000g for 10 min. To each pellet, 100 ll of dimethylsulfoxide (DMSO) was added to dissolve the formazan for 10 min. Absorbance was measured on ELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain sample signals. Results Construction of the recombinant P. pastoris Using a custom designed primer pair, the desired fragment of human IL-7 gene was ampli?ed by PCR (see Materials and meth-

Y. Luo et al. / Protein Expression and Puri?cation 63 (2009) 1–4

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ods). The ampli?ed fragment was con?rmed to be hIL-7 gene by sequence analysis. The IL-7 gene was cloned into pPICZaB, where it is under the control of oxidase I promoter from the vector. The resulting plasmid, pPICZaB-hIL-7, was transformed into P. pastoris and plated on YPD medium containing low Zeocin (100 lg/ml). Fifteen colonies grown on the low Zeocin selection plate were replated on YPD plates containing high Zeocin (500 lg/ml). Following 48 h of incubation at 30 °C, three large colonies were selected for further analysis. Small-scale fermentation and time course expression study Three isolates were induced in 250 ml cultures for 4 days, and supernatant samples were collected every 12 h for analysis by SDS–PAGE and Western blot. All three clones had similar expression patterns and results from one of the representative are shown. As shown in Fig. 1A, secreted rhIL-7 could be observed on Coomassie blue R250 stained gel as a 17 kDa band. Western blot analysis of the same samples (Fig. 1B) showed a progressive accumulation of the rhIL-7, starting 12 h post induction and reached the highest peak by 48 h. Prolonged induction (>48 h) resulted in decreased amount of total rhIL-7 in the culture supernatant, suggesting possible breakdown of the protein. Large-scale expression and puri?cation of the rhIL-7 Biological potency and stability of the rhIL-7 One of the three isolates was grown on a 30-L reactor and induced with methanol for 48 h. Culture medium was centrifuged and the supernatant was collected. Total protein concentration in the supernatant was 2.73 g/L as estimated by Invitrogen Quibt ?uorometer while the rhIL-7 protein was estimated to be 55 mg/ L according to Western blot analysis. The rhIL-7 was puri?ed from the supernatant by passing through ion exchange (SP Sepharose) chromatography (see Materials and methods). SDS–PAGE analysis of the puri?ed sample showed single band slightly above 17 kDa, which is consistent with the molecular weight of human IL-7. Purity of the rhIL-7 reached about 95% as revealed by SDS–PAGE (lane 3, Fig. 2). Out of the 24 L culture supernatant, we obtained total 0.84 g of puri?ed rhIL-7, representing 63% recovery rate of the total rhIL-7 from the culture supernatant. To address the speci?c biological activity of the puri?ed rhIL-7, MTT assay was conducted. Different concentrations of the puri?ed rhIL-7 (1 pg/ml–100 ng/ml) were added into 2E8 cells (2 ? 105) grown on 96-well plates. After 48 h of incubation, cell proliferation was determined (see Materials and methods). As Fig. 3 shows, addition of as low as 10 pg/ml rhIL-7 stimulated the growth of 2E8 cells, corresponding to a speci?c activity of 106 IU/mg. This activity is comparable to the values reported for the protein derived from E. coli expression system [19] and the commercially available rhIL-7 from E. coli. Puri?ed rhIL-7 showed no signi?cant loss in its activity when stored for more than 4 months at ?20 °C in PBS buffer (pH 7.0). Discussion The role of cytokine IL-7 has been studied for the past 20 years. It plays a unique role in T-cell development, survival, and establishment of immunological memory. A recent study demonstrated

Fig. 2. Puri?cation of rhIL-7. SDS–PAGE analysis of puri?ed rhIL-7 samples from Pichia pastoris. Lane1, molecular weight marker; lane 2, culture supernatant after 48 h of induction; lane3, elution fraction at 2 M NaCl in PBS (5 lg protein).

Fig. 1. Small-scale expression and time course study of rhIL-7 expression in Pichia pastoris X-33. (A) SDS–PAGE analysis of culture supernatant during small-scale expression of rhIL-7. P. pastori X-33 transformed with pPICZaB-hIL-7 was induced with methanol for 96 h. Culture supernatant (10 ll) was taken at the indicated time points and subjected to SDS–PAGE analysis. (B) Western Blot analysis for rhIL-7 protein. A sister SDS–PAGE gel as shown in (A) was transferred onto a membrane and blotted with anti-hIL-7 monoclonal antibody.

Fig. 3. Effect of puri?ed rhIL-7 on the proliferation of 2E8 cells. Indicated amounts of puri?ed rhIL-7 were supplemented into the 96-well plates containing 2 ? 105 of 2E8 cells. Following 48 h of incubation, cell proliferation was measured by MTT assay. The data represents means ± error standard of three independent experiments.

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that it is also a vital factor in T-cells to support cell metabolism and cell survival through promoting Glut1 traf?cking and glucose uptake via STAT5-mediated activation of Akt [14]. Its promising value in clinical applications is highlighted by the potential implementation of IL-7 to treat various diseases, such as bone marrow and organ transplantation, cancer immunotherapy, and infectious diseases [18–20]. These potential applications require large-scale quantities of puri?ed recombinant protein. Currently, rhIL-7 is mainly produced from prokaryotic expression system by the use of E. coli expression system. A major drawback of using E. coli is the formation of inclusion bodies which require refolding, thus resulting in a low puri?cation yield. Also, contamination by E. coli derived pyrogenic components is of significant concern for clinical applications. In order to ?nd an ef?cient way to produce large amount of bioactive rhIL-7 for both structural and clinical studies, we used methylotrophic yeast, P. pastoris, as an expression system. This system has become increasingly popular in recent years due to its ease of genetic manipulation and growth to a high cell density using simple minimal medium, resulting in high yields. Furthermore, pyogenic contamination is no longer a concern. In fact, many pharmaceutically important proteins have successfully been produced using his system for clinical applications [21]. This study demonstrates the suitability of P. pastoris to process and secrete intact rhIL-7 that retains the properties of the native protein. With a simple one-step puri?cation by the SP Sepharose FF cation-exchange, we have obtained 35 mg/L protein compared to 10 mg/L product puri?ed from E. coli inclusion bodies [19], with much of the toxicity issue eliminated since it is a secreted extracellular protein. Bioactivity assays have also shown that 10 pg/ml recombinant protein is suf?cient to stimulate cell proliferation, indicating a similar level of bioactivity as the commercial products derived from E. coli. In conclusion, this paper describes a novel and highly ef?cient way of producing large quantities of functional rhIL-7, and this system may not only facilitate further studies of its structure–function relationship as well as clinical applications, but also can allow possible large-scale production of biologically active rhIL-7. Acknowledgments This work was supported in part by Funds from the National Science Foundation of China (30670457), Guangzhou Administration of Science and Technology (2007Z2-E4021 and 2005Z3-C7181), Guangzhou Economic and Technological Development District matching funds (2007Ss-P059) and the National 973 program of China (2007CB914301, 2006CB910202 and 2004CB720102). References
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