High-yield soluble expression of recombinant human ... 12- Xue Zhu-rec6sept2014_ac 20... ·...

Romanian Biotechnological Letters Vol. 20, No. 1, 2015 Copyright © 2015 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 20, No. 1, 2015 10114

High-yield soluble expression of recombinant human interleukin-1β in Escherichia coli

Received for publication, September 06, 2014 Accepted, January 14, 2015

XUE ZHU1, AIPING LIU2*, KAI ZHANG1, KE WANG1*, YUN GAO1, FANFAN ZHOU3, LING ZHU4 1 Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of

Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province, China;

2 Wuxi Environment Science and Engineering Research Center, Wuxi City College International Education School, Wuxi 214000, Jiangsu Province, China;

3 Faculty of Pharmacy, University of Sydney, NSW 2006, Australia; 4 Save Sight Institute, University of Sydney, NSW 2000, Australia * Corresponding author: Email: [email protected];[email protected]

Abstract

The pro-inflammatory cytokine interleukin-1β (IL-1β) is a key mediator of inflammation and is implicated in the pathogenesis of diverse disease states. Due to its strong immunomodulatory effects, IL-1β may be widely used in basic and clinical research. Previously, many studies reported that IL-1β could be produced in Escherichia coli (E. coli), however, low-yield and insoluble inclusion bodies in production are the major bottlenecks. In this study, we described a novel and efficient method to express functional recombinant human IL-1β (rhIL-1β) in E. coli. The human IL-1β gene was codon optimized and then expressed by single protein production (SPP) expression system. The results indicated that rhIL-1β was high-level soluble expressed, and upon purification by affinity and ion-exchange chromatography, the final yield was about 247 mg/L in shake flask with biological activity of stimulating the production of interleukin 2 (IL-2) in EL4 cells. Our study may provide a sound strategy for large-scale production of functional rhIL-1β from E. coli for further studies in the future.

Keywords: Recombinant human IL-1β, Codon optimization, Single protein production system, Escherichia coli

1. Introduction

Interleukin-1β (IL-1β) is a biologically active small protein molecule known as “multi-functional” pro-inflammatory cytokine which is produced by a number of different cell types and affecting nearly all cell types, either alone or in combination with other cytokines (1, 2). It participates in a variety of inflammatory diseases, including gouty arthritis, type 1 diabetes and the NALP3-associated disorders (3, 4). Nowadays, blocking IL-1β is the standard of therapy for “auto-inflammatory” diseases (5, 6). Numerous studies have highlighted the important role of IL-1β in basic and clinical research, however, large-scale production of functional recombinant human IL-1β (rhIL-1β) is imperative.

The active mature human IL-1, which apparently processed form 31kDa precursors to about 17kDa, are159 and 153 residues long and are respectively designated as IL-1α and IL-1β (7). The first cloning and expression of rhIL-1β in Escherichia coli (E. coli) was reported in the early 1980s. Heterogonous expression of recombinant proteins in E. coli is one


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of the most cost-efficient methods to provide sufficient quantities for functional studies, but there is still no guarantee that a recombinant gene product will accumulate in E. coli at high levels in a biologically active form (8). Low-yield and insoluble inclusion bodies in production are the major bottlenecks in traditional expression of rhIL-1β in E. coli, which become a serious impediment to the process of further IL-1β functional researches.

In this study, we described a high-output expression of soluble rhIL-1β in E. coli. Human IL-1β gene was codon-optimizedbased on the codon preference of E. coli without changing the amino acid sequence, then expressed by single protein production (SPP) system. This system provides two huge advantages for high-yield heterologous protein expression. The pCold vector provided in SPP system expresses the protein in a low-temperature condition, which could extremely reduce misfolding and segregation into insoluble inclusion bodies, and increase the solubility and biological activity of the recombinant protein (9). On the other hand, a single protein of interest is produced by co-expression with mRNA interferaseMazF in SPP system (9). MazF specifically cleaves cellular mRNAs at ACA nucleotide sequences, which means the protein of interest (with non-MazF-cleavable sequences) is produced virtually in the absence of background cellular protein synthesis (10). To the best of our knowledge, this is the first report on the high-yield production of soluble rhIL-1β by SPP system in E. coli. 2. Materials and methods 2.1. Materials and Reagents

SPP system is obtained from Dr. Li Wenjing’s laboratory and which includes pCold and pMazF vectors. E. coliBL21 (DE3) (conserved by our laboratory) was used for protein expression. Restriction enzymes and T4 ligase were purchased from Takara (Otsu, Japan). RPMI medium 1640, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco (Grand Island, NY, USA). Humanrecombinant IL-2 was purchased from ZeptoMetrix (Buffalo, NY, USA). Ampicillin and chloramphenicol were purchased from Sangon (Shanghai, China). IPTG (isopropy-β-D-thiogalactoside) was purchased from Sigma Chemical Co. (St Louis, USA). HisTrap HP, HiTrap Q and AKTA purifier system were purchased from GE Healthcare (New Jersey, USA).

2.2. Construction of the Expression Plasmid

Based on the cDNA sequence of human IL-1β (NM_000576.2), a synthetic coding sequence has been designed considering the optimal codon usage for E. coli. In addition, ACA sequences in the cDNA were replaced to non-MazF-cleavable sequences without changing amino acid sequences. The sequence-optimized cDNA was integrated in frame the vector pCold using XbaI/NdeI digestion. The recombinant plasmid containing human IL-1β cassette in frame was confirmed by restriction endonuclease and DNA sequencing.

2.3. Expression of rhIL-1β

The expression plasmid pCold/rhIL-1β and the helper plasmid pMazF were simultaneously transformed into the host E. coli strain BL21 (DE3). Cell cultures were induced by the addition of 1 mM IPTG at 15ºC when OD600 reached 0.5 and cells were harvested after 24 h. For evaluation of protein expression, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out. Protein concentration was assessed by Bradford method (11).

XUE ZHU, AIPING LIU, KAI ZHANG, KE WANG, YUN GAO, FANFAN ZHOU, LING ZHU


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2.4. Purification of rhIL-1β The bacterial cells were re-suspended in binding buffer (pH 8.0) containing 10 mM

imidazole and ultrasonic for 30 min. The lysate was centrifuged and the supernatant of E. coli lysate containing rhIL-1β was passed through pre-equilibrated HisTrap HP column. The column was washed with binding buffer, after that the His-tagged proteins was eluted with a gradient of imidazole ranged from 100-500 mM and all the flow-through was collected. The elute fractions containing the majority of the target protein was further purified with a Q Sepharose FF column (GE Healthcare, Sweden) and eluted with linear gradient of 0-1.0 M NaCl in phosphate buffered saline (PBS). The fractions were monitored at OD280 using the optical unit of a Fast protein liquid chromatography system (AKTAprime plus, GE Healthcare, Sweden). A putative peak containing the rhIL-1β was identified and collected for analysis. The purified protein was further evaluated by SDS-PAGE and western blotting analysis.

2.5. Western blotting analysis

Protein concentration was determined by the Bradford method(11). Cell lysates were electrophoresed on a 15% SDS polyacrylamide gel and transferred onto PVDF membrane. After blocking with 5% bovine serum albumin (BSA) in the mixture of Tris-Buffered Saline and Tween-20 (TBST) for 1 h, membranes were incubated with primary antibody overnight and followed by incubation with secondary antibody for 1 h at room temperature. Protein bands were visualized using the ECL assay kit (Beyotime, Nantong, China). The density of each band was normalized to the expression of β-actin.

2.6. Cell culture

Mouse T cell leukemia cell line EL4 and mouse cytotoxic T lymphocyte (CTL) cell line CTLL-2, were obtained from Shanghai Cell Bank, Chinese Academy of Sciences. CTLL-2 cells were cultured in RPMI 1640 medium supplemented with 100 IU/ml human recombinant IL-2, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. EL4 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution. Both cell lines were maintained at 37ºC in a humidified atmosphere containing 5% CO2.

2.7. Bioassay for rhIL-1β

Thebiological activity of rhIL-1β was determined by its ability of stimulating the production of interleukin 2 (IL-2) in EL4 cells, which then could maintain the survival of IL-2-dependent CTLL-2 cell line [12]. Murine T-cell line EL4 cells (2×104 cells/well) and CTLL-2 cells (4×104 cells/well) were co-seeded into 96-well culture plates with RPMI 1640 containing 10% fetal bovine serum. After IL-2 starved for 24 h, cells were treated with various concentrations of rhIL-1β (0, 1, 2, 5, 10, 25 and 50 nM) for 24 h. At the end of time points, cell proliferation was measured by MTT assay as mentioned before (13). Cell proliferation was expressed as a percentage of the control. The experiment was done in triplicate repeats and the results are indicative of three independent studies.

2.8. Statistical analysis

Biostatistical analyses were completed by the SPSS 16.0 software package (Chicago, IL, USA). All experiments were repeated three times. Results of multiple experiments were expressed as mean ± SD. A P value less than 0.05 was accepted as statistically significant.



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3. Results 3.1. Expression of rhIL-1β influenced by codon optimization

Species-specific variations in codon usage are one of the major causes affecting the expression level and solubility of target protein (14). Previous studies have reported that heterologous expression of some proteins in E. coli can be improved by altering the codon to better match the preference of the host (15). To investigate the effect of codon optimization on the expression of rhIL-1β in E. coli, the native and codon-optimized human IL-1β genes were respectively inserted into pCold vector and expressed in E. coli BL21(DE3) under the control of the inducible cold-shock protein cspA promoter and terminator (Fig. 1A). The expression level was scored by the visualization of a band of the correct size by SDS-PAGE. The results showed that the expression level of the codon-optimized gene was about 2.4 fold higher than that of the native gene. It is obvious that heterologous expression of human IL-1β in E. coli can be significantly improved by altering codon preference.

3.2. Expression and solubility of rhIL-1β using SPP system

MazF is a toxin encoded by the E. coli chromosome and it can specifically cleaves cellular mRNAs at ACA nucleotide sequences as an mRNA interferase(9, 10). For SPP system, the protein of interest from an mRNA without ACA sequences could be high-level produced (10). The schematic diagram of SPP system is showed in Figure 2A. The codon-optimized ACA-less human IL-1β gene was co-expressed with the MazF gene in SPP system. The results clearly demonstrated that rhIL-1β was high-level expressed at a low background cellular protein synthesis upon MazF induction (Fig. 2B). The yield of rhIL-1β was approach to 45% of total cellular proteins.In addition, expression at low temperature could significantly improve the solubility of recombinant proteins [16]. In SPP system, a promotor of the cold-shock protein cspA is utilized in the pCold vector to express the target protein under cool condition. In this study, we observed efficient soluble expression of rhIL-1β by using pCold vector (Fig. 2C). Further experiment indicated that over 90% of rhIL-1β was expressed in a soluble form.

A B

Figure 1. Influence of codon optimization on recombinant human IL-1β expression in E. coli.A: Upper panel, Sequence comparison between native and codon-optimized human IL-1β gene; Lower panel, Schematic representation of the expression vector. B: Expression level comparison between native and codon-optimized human IL-1β gene in E. coliBL21(DE3). Lane M: molecular weight



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marker, Lane 1: empty vector control, Lane 2: Expression from vector with native human IL-1β gene, Lane 3: Expression from vector with codon-optimized human IL-1βgene. 3.3. Purification of rhIL-1β using chromatography

As showed in Figure 1A, rhIL-1β was expressed as a soluble form with His-tag. The target protein was purified by affinity chromatography using HisTrap HP (Ni2+-NTA) column and ion-exchange chromatography using HiTrapQ column. The protein mixture was first purified by HisTrap HP column. Eluted rhIL-1β fractions were pooled, dialyzed and cleaved by Factor Xa protease to remove the His-tag. Then the protein was further purified by HiTrap Q column using Fast protein liquid chromatography (FPLC). The purity of the purified soluble rhIL-1β was analyzed by SDS-PAGE through staining with Coomassie Brilliant blue (Fig. 3). The purification of rhIL-1β under native conditions produced single band of ~17 kDa protein with high purity (>95 %) (Data not shown). Immunoblot analysis with anti-human IL-1β antibody confirmed the identity of the purified protein which has the molecular weight of 17 kDa (Fig.4). In addition, the purified rhIL-1β achieved a high yield of about 247 mg/L cell culture after purification in the present study, which is the highest to date.

A

B C

Figure 2. Expression and solubility of recombinant human IL-1β in E.coli using SPP system.A: The schematic diagram of the SPP system. B: Expression analysis of rhIL-1β by SDS-PAGE. Lane M: molecular weight marker, Lane 1: Without co-expression with MazF, Lane 2: With co-expression with MazF. C: Solubility analysis of rhIL-1β by SDS-PAGE. M: molecular weight marker, Lane 1: Crude cell lysate, Line 2: Soluble fraction, Line 3: Insoluble fraction.



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Figure 3. Purification of recombinant human IL-1β by using chromatography.Lane M: molecular weight marker, Lane 1: Crude cell lysate, Lane 2: The intermediate product of rhIL-1β purified by HisTrap HP column, Lane 3: The final product of rhIL-1β purified by HiTrap Q column.

Figure 4. Western blot analysis of rhIL-1β by anti-hIL-1β antibody.Lane 1: Control group without rhIL-1β, Lane 2: The purified rhIL-1β protein.

3.4. Functional characterization of rhIL-1β

The bioactivity of rhIL-1β was measured by its ability of stimulating the production of interleukin 2 (IL-2) in EL4 cells, which could then promote cell survival of IL-2-dependent CTLL-2 cells. Cell viability of CTLL-2 cells treated with the indicated protein was evaluated with MTT assay as described previously (12). As shown in Figure 5, purified rhIL-1β from our system exhibited a remarkably proliferative effect on the growth of CTLL-2 cells, which was higher than that of the commercially available rhIL-1β from E. coli. In addition, purified rhIL-1β showed no significant loss in its bioactivity when stored for more than 4 months at –20ºC.



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Figure 5. Bioactivity assay of purified rhIL-1β. Cells were pretreated with various concentrations of rhIL-1β (0, 1, 2, 5, 10, 25 and 50 nM) for 24 h. After incubation, cell viability was evaluated by MTT assay. The results are showed as Mean ± SEM of three experiments and each included triplicate sets. Control: commercially available rhIL-1β; rhIL-1β: rhIL-1β from SPP system. a: p< 0.05 Starving group vs. IL-2; b: p< 0.05 Control rhIL-1βvs. Starving group; c: p< 0.05 rhIL-1β vs. Control rhIL-1β.

4. Discussion

As recombinant proteins have been widely used in industry, medical and basic researches, high yield of soluble biologically active recombinant proteins has broad application prospects. For decades, a series of expression systems have been used for the production of heterologous proteins, including Escherichia coli, Pichiapastoris, the baculovirus expression system and mammalian cells (17-19). E. coli is the most widely used expression system over the othersdue to its two great advantages, the speed of producing overexpressing strains and the ease and cheapness of large-scale production (20). Different cultivation and molecular techniques have been developed to increase the yield of recombinant proteins. However, high-level production of functional proteins in E. coliis still sometimes challenging. Not every protein can be efficiently produced due to the unique structural features of the protein, its folding pathways and its degradation by host cell proteases (21). The aim of this study was to produce high yield of soluble and bioactive recombinant human IL-1β with less cost and simple purification steps in the E. coli expression system.

Biased codon usage is a major problem that diminishes the efficiency of heterologous protein production in E. coli. The codon bias of the gene to be expressed differs significantly form that used by E. coli have been shown to cause translational errors such as stalling, termination, amino acid substitution, frame shifting as well as lower levels of protein expression (15). The current technology has enabled the cost-effective synthesis of complete genes, and it is convenient to obtain codon-optimized and mRNA secondary structure-free sequences from commercial sources (22). To improve the production of rhIL-1β in E. coli, we carried out the codon optimization of rhIL-1β gene according to the specific codon usage of E. coli (codon usage database from http://www.kazusa.or.jp/codon), in which, ACA sequences



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in cDNA were additionally replaced to non-MazF-cleavable sequences. As shown in our study, the expression level of rhIL-1β was enhanced about 2.4-fold by using sequence-optimized constructs. Furthermore, the optimized gene was expressed by SPP system,which was designed to produce only a single protein of interest in living E. coli. In SPP system, the protein of interest was co-expressed with an mRNA interferaseMazF, since MazF induction in E. coli causes degradation of almost all cellular mRNAs, target protein can be expressed efficiently with a very high signal-to-noise ratio (9). It could be clearly observed in Figure 2B that co-expression with MazF significantly improved the relative amount of rhIL-1β in total cellular protein production.

Under overexpression conditions, proteins frequently accumulate as insoluble aggregates (inclusion bodies) in E. coli expression system. Several experimental approaches have been developed to overcome this problem and one of the effective methods is to lower the growth temperature of induced cultures (23). The pCold vector in SPP system contains the promoter of the cold shock-inducible gene for the major cold shock protein CspA, allowing a very high level of expression of a cloned gene upon cold shock. Low-temperature induction improves not only the yield of the expression but also provides the advantages of better protein folding and increases the solubility of the protein (Figure 2C). Then, with a hexahistidine (His6) tag, rhIL-1β was easily purified by affinity and ion-exchangechromatography, and the final yield was approximate 247 mg/L in shake flask after purification. The production of IL-2, by the stimulation of IL-1β in a culturedEL4 murine T-cell line (12), was then measured by testing cell proliferation of IL-2-dependent mouse cytotoxic T-cell line (CTLL-2) by MTT assay. Bioactivity assay showed that the biological activity of purified rhIL-1β from SPP system was higher than that of the commercially available rhIL-1β from E. coli expression system.

In conclusion, we described a cost-effective method for the large-scale production of soluble and functional rhIL-1β in E. coli. Our study reported here may provide an important tool for further studies in the future. 5. Acknowledgments

This work was supported by grants from National Natural Science Foundation (81300787), the Natural Science Foundation of Jiangsu Province (BK2011168, BK2012105, BK20141103) and Youth Foundation of Jiangsu Institute of Nuclear Medicine (QN201111). References

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