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Efficient overexpression of human interleukin-6 in Escherichia coli using nanoluciferase as a fusion partner
Process Biochemistry 50 (2015) 1618–1622
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Efficient overexpression of human interleukin-6 in Escherichia coli using nanoluciferase as a fusion partner Ben-Jun Ji a,b , Ge Song b , Zhou Zhang a , Zhan-Yun Guo b,∗ a b
College of Life Sciences, Shanghai Normal University, Shanghai, China Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China
a r t i c l e
i n f o
Article history: Received 26 March 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online 14 June 2015 Keywords: Fusion partner E. coli NanoLuc Interleukin-6
a b s t r a c t Escherichia coli is widely used to produce recombinant proteins. For soluble overexpression, fusion technology has been developed using different fusion partners. In the present work, we used a newly developed nanoluciferase (NanoLuc) as a novel fusion partner for soluble overexpression of the aggregation-prone interleukin-6 (IL6). Soluble 6×His-NanoLuc-IL6 fusion protein was efficiently overexpressed in E. coli at a yield of approximately 60 mg per liter of culture broth after purification. The fusion partner was removed by enterokinase digestion and monomeric mature IL6 was obtained at a final yield of approximately 15 mg per liter of culture broth. The recombinant IL6 protein was fully active in the receptor activation assay, with a measured EC50 value of 117 ± 10 pM (n = 3). Thus, the novel NanoLucfusion approach was efficient for the soluble overexpression of active IL6 protein in E. coli and could be applied to other aggregation-prone proteins in future studies. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli is widely used to produce recombinant proteins in both the laboratory and industry [1–3]. In past decades, numerous heterologous proteins have been successfully overexpressed in E. coli. However, some proteins are prone to form insoluble inclusion bodies when overexpressed in E. coli and are hard to be renatured in vitro. For soluble overexpression of these aggregationprone proteins in E. coli, fusion technology has been developed, in which a highly soluble fusion partner protein [4–7], such as glutathione S-transferase (GST), maltose-binding protein (MBP), SUMO, NusA, thioredoxin (Trx), or ubiquitin is covalently fused to the target protein, typically at the N-terminus. Unfortunately, the solubilizing effect of these fusion partners varies significantly with different target proteins [8,9]. In a recent work, we found that fusion of a newly developed small nanoluciferase (NanoLuc, 19 kDa, 171 amino acids) significantly improved the soluble overexpression of the aggregation-prone human leukemia inhibitory factor (LIF) in E. coli [10], suggesting that NanoLuc might be a novel fusion partner. Interleukin-6 (IL6), also named interferon-ˇ2 (IFNB2), B-cell stimulatory factor 2 (BSF-2), hybridoma growth factor (HGF), and hepatocyte stimulating factor (HSF) in early references, is a well defined pro- and anti-inflammatory cytokine/myokine
that is involved in nearly all pathophysiological states by binding and activating a heterodimeric IL6R/gp130 receptor [11–13]. Mature human IL6 protein comprises 184 amino acids and forms a four-helix bundle structure with two disulfide bonds [14,15]. Overexpression of IL6 in E. coli has been attempted by several laboratories [16–25]. However, the yield of soluble active IL6 was low because of its high aggregation propensity, although different fusion partners were used. In the present work, we applied the novel NanoLuc-fusion approach to IL6 and showed it to be an efficient approach for overexpression of fully active IL6 in E. coli. 2. Materials and methods 2.1. DNA manipulation The codon-optimized nucleotide sequence of mature human IL6 was chemically synthesized and ligated into the previously generated pNLuc vector [10], after digestion with restriction enzymes EcoRI and HindIII, resulting in the expression construct pNLuc/IL6. The coding sequence of human IL6 was confirmed by DNA sequencing. 2.2. Overexpression and purification of 6×His-NanoLuc-IL6
∗ Corresponding author. Tel.: +86 21 65988634; fax: +86 21 65988403. E-mail address: [email protected] (Z.-Y. Guo). http://dx.doi.org/10.1016/j.procbio.2015.06.008 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
The expression construct pNLuc/IL6 was transformed into E. coli strain BL21(DE3) and transformants were cultured in liquid LB medium to OD600 nm = 1.0 at 37 ◦ C. Thereafter, the inducer isopropyl
B.-J. Ji et al. / Process Biochemistry 50 (2015) 1618–1622
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Fig. 1. Overexpression and purification of 6×His-NanoLuc-IL6. (A) Flowchart of the purification and maturation process. (B) Induced overexpression analyzed by nonreducing SDS-PAGE. Lane (−), total cell lysate before IPTG induction (from 30 l of culture broth); lane (+), total cell lysate after IPTG induction (from 30 l of culture broth). (C) Solubility analyzed by non-reducing SDS-PAGE. Lane s, supernatant after sonication (1.0 l of sonication supernatant); lane p, pellet after sonication (from 1.0 l of sonication mixture). (D) Ni2+ column purification analyzed by non-reducing SDS-PAGE. Lane f, flowthrough fraction (1.0 l of flowthrough); lane w, fraction washed by 30 mM imidazole (1.0 l of wash fraction); lane e, fraction eluted by 250 mM imidazole (1.0 l of eluent). (E) Gel filtration purification analyzed by non-reducing SDS-PAGE. Lane p1, the first peak eluted from the gel filtration column (1.0 l of eluent); lane p2, the second peak eluted from the gel filtration column (1.0 l of eluent). The band corresponding to 6×His-NanoLuc-IL6 is indicated by an asterisk (*).
-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM and the cells were continuously cultured at 17 ◦ C for ∼13 h with gentle shaking (100 rpm). Subsequently, the E. coli cells were harvested by centrifugation (6000 × g, 5 min), resuspended in lysis buffer (20 mM phosphate buffer, pH 7.5, 0.5 M NaCl), and lysed by sonication. After centrifugation (12,000 × g, 30 min), the supernatant was applied to an immobilized metal ion affinity chromatography (Ni2+ column) and the bound 6×HisNanoLuc-IL6 protein was eluted by lysis buffer containing 250 mM imidazole. The 6×His-NanoLuc-IL6 fraction eluted from the Ni2+ column was subsequently loaded onto a gel filtration column (TSKgel G2000SWXL, 7.8 mm × 300 mm, Sigma Aldrich, St. Louis, MO, USA), eluted with 20 mM Tris–Cl buffer (pH 8.5), manually collected and analyzed by non-reducing SDS-PAGE.
2.3. Enterokinase cleavage of 6×His-NanoLuc-IL6 The 6×His-NanoLuc-IL6 fraction eluted from the gel filtration column was subjected to enterokinase digestion. CaCl2 was added to a final concentration of 2.0 mM and enterokinase (New England Biolabs, Ipswich, MA, USA) was added to a final concentration of ∼2 ng/ml. Digestion was carried out at 25 ◦ C overnight. Thereafter, the digestion mixture was loaded onto a DEAE ion-exchange column (TSKgel DEAE-5PW, 7.5 mm × 75 mm, from Sigma Aldrich). The fractions eluted by a gradient of sodium chloride (in 20 mM phosphate buffer, pH 7.5) were manually collected and analyzed by non-reducing SDS-PAGE.
2.4. Circular dichroism measurement The concentration of the purified mature IL6 was quantified by the 2,2 -bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as standard. Its final concentration was adjusted to 0.1 mg/ml for circular dichroism measurement, which was performed on a Jasco-715 spectrometer at room temperature. The spectrum was scanned from 180 to 250 nm using a quartz cuvette with 1.0 mm path length.
2.5. Biological activity assay of the recombinant IL6 The biological activity of the recombinant IL6 was assayed as its receptor activation potency using a STAT3-response sis-inducible element (SIE)-controlled NanoLuc as a reporter [10]. The reporter construct pNL1.2/SIE was transiently transfected into HEK293T cells. Next day, the transfected cells were typsinized, seeded into a 96-well plate, and continuously cultured to approximately 90% confluence. Thereafter, the medium was removed and the assay solution (serum-free DMEM medium plus 1% bovine serum albumin), containing different concentrations of mature IL6, was added (100 l/well) and the cells were continuously cultured at 37 ◦ C for 7 h. Subsequently, the assay solution was removed and the cells were lysed by adding lysis solution (100 l/well, from Promega, Madison, WI, USA). The cell lysate was then transferred to a white, opaque 96-well plate (50 l/well) and the bioluminescence was immediately measured on a SpectroMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA), after addition of the diluted substrate (50 l/well, from Promega). The measured data were expressed as mean ± SE and fitted to a sigmoidal curve using the SigmaPlot10.0 software. 3. Results and discussion 3.1. Overexpression and purification of 6×His-NanoLuc-IL6 in E. coli Overexpression of soluble IL6 in E. coli is difficult because of its high aggregation propensity [16–25]. To increase solubility of IL6 in E. coli, we tried the novel NanoLuc fusion partner. The mature human IL6 gene was chemically synthesized using E. coli-optimized codons and ligated into a pNLuc vector, which carries the coding sequence of 6×His-NanoLuc upstream of the IL6 gene. The condon usage of the synthetic human IL6 gene was manually harmonized to make it consistent with the statistic codon usage of E. coli (http://www.kazusa.or.jp/java/codon table java/). The nucleotide and amino acid sequence of 6×His-NanoLuc-IL6 fusion protein is shown in supplementary Fig. S1. The overexpression, purification
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and maturation process of 6×His-NanoLuc-IL6 is shown in Fig. 1A. After the expression construct pNLuc/IL6 was transformed into E. coli strain BL21(DE3), transformants were cultured in liquid LB medium and induced by IPTG at low temperature (17 ◦ C). After induction, a strong protein band (indicated by an asterisk) with a molecular weight of ∼40 kDa appeared on non-reducing SDSPAGE (Fig. 1B), suggesting that monomeric 6×His-NanoLuc-IL6 (theoretical molecular weight of 42.8 kDa) was efficiently overexpressed after IPTG induction. We wanted to detect whether the fused or mature IL6 formed oligomers through intermolecular disulfide crosslinking in E. coli and in the later purification processes, thus we used the non-reducing SDS-PAGE throughout the experimental procedures, except when stated otherwise. After the E. coli cells were lysed by sonication, the fusion protein was predominantly present in the supernatant, as analyzed by nonreducing SDS-PAGE (Fig. 1C), suggesting that the overexpressed 6×His-NanoLuc-IL6 was soluble. Thus, the NanoLuc fusion partner significantly increased solubility of the aggregation-prone IL6 protein in E. coli. The soluble 6×His-NanoLuc-IL6 was initially purified by an immobilized metal-ion affinity chromatography based on the N-terminal 6×His-tag. As analyzed by non-reducing SDS-PAGE (Fig. 1D), monomeric 6×His-NanoLuc-IL6 was a major band in the eluted fraction, although some minor bands were also present. After purified by Ni2+ column, typically ∼100 mg of monomeric 6×HisNanoLuc-IL6 could be obtained from one liter of E. coli culture broth as analyzed by non-reducing SDS-PAGE and densitometry using BSA as a standard. Thereafter, 6×His-NanoLuc-IL6 was further purified by gel filtration and two peaks were eluted. As analyzed by non-reducing SDS-PAGE (Fig. 1E), the first peak (p1) was almost homogenous monomeric 6×His-NanoLuc-IL6 and the second peak (p2) contained other smaller proteins. After two-step purification, approximately 60 mg of monomeric 6×His-NanoLuc-IL6 protein was typically obtained from one liter of E. coli culture broth as analyzed by non-reducing SDS-PAGE and densitometry. Thus, soluble, monomeric 6×His-NanoLuc-IL6 fusion protein could be efficiently overexpressed in E. coli using the novel NanoLuc-fusion approach. Supplementary Fig. S1 related to this article can be found, in the online version, at doi:10.1016/j.procbio.2015.06.008 3.2. Preparation and characterization of mature IL6 To obtain the mature IL6 protein, the purified 6×His-NanoLucIL6 was treated with enterokinase, which cleaves the fusion protein at the C-terminus of the DDDDK sequence (Fig. S1). As analyzed by non-reducing SDS-PAGE (Fig. 2A), after enterokinase digestion, the band corresponding to 6×His-NanoLuc-IL6 (42.8 kDa) disappeared and two smaller bands appeared, suggesting that enterokinase had cleaved the fusion protein efficiently. To separate the cleaved 6×His-NanoLuc fusion partner from the mature IL6, the digestion mixture was loaded onto a DEAE ion-exchange column, because the cleaved 6×His-NanoLuc and the mature IL6 have opposite charges. The positively charged mature IL6 would flow through the column, while the negatively charged 6×His-NanoLuc would be bound by the positively charged DEAE resin, and would only be eluted by a salt gradient. As analyzed by non-reducing SDS-PAGE (Fig. 2B), the cleaved 6×His-NanoLuc and the mature IL6 were well separated by the ion-exchange chromatography, because each fraction contained a single protein band. The mature IL6 (theoretical value of 21.2 kDa) had a slightly larger apparent molecular weight on the non-reducing SDS-PAGE than the cleaved 6×His-NanoLuc (theoretical value of 21.6 kDa). After treatment by the reducing reagent dithiothreitol (DTT), which breaks disulfide bonds, the mobility of the mature IL6 decreased slightly on SDS-PAGE (Fig. 2C), suggesting that the mature IL6 formed intramolecular disulfide bonds that rendered the polypeptide chain more compact and thus ran faster on SDS-PAGE. As analyzed by circular dichroism spectroscopy
Fig. 2. Maturation and characterization of the recombinant IL6. (A) Enterokinase digestion analyzed by non-reducing SDS-PAGE. Lane (−), fusion protein before enterokinase digestion; lane (+), fusion protein after enterokinase digestion. The band corresponding to 6×His-NanoLuc-IL6 is indicated by an asterisk (*); the band corresponding to mature IL6 is indicated by a double asterisk (**); the band corresponding to the cleaved 6×His-NanoLuc is indicated by a hash (#). (B) Ionexchange chromatography purification analyzed by non-reducing SDS-PAGE. Lane f, flowthrough fraction; lane e, fraction eluted by a gradient of sodium chloride. (C) SDS-PAGE analysis of the mature IL6 before (−) and after (+) DTT treatment. (D) Circular dichroism spectrum of the mature IL6.
Fig. 3. Biological activity assay of mature IL6 using a STAT3-response NanoLuc reporter. The measured bioluminescence data are expressed as mean ± SE (n = 3) and fitted to a sigmoidal curve using SigmaPlot 10.0 software.
B.-J. Ji et al. / Process Biochemistry 50 (2015) 1618–1622 Table 1 Summary of the recombinant expression of IL6 in E. coli. Approach
Final yielda
Reference
Soluble expression of 6×His-NanoLuc-tagged IL6; tag removed by enterokinase after purification. Inclusion body formation of 6×His-tagged IL6; in vitro on-column refolding after solubilization; without tag removal step. Soluble expression of 6×His-tagged IL6 through coexpression of chaperones; without purification and tag removal steps; Solubilizing effect of GST was not good. Inclusion body formation of 6×His-tagged IL6; in vitro refolding by dialysis; without tag removal step. NusA, BMP, Trx, ubiquitin tags were tested and NusA had the best solubilizing effect; without purification and tag removal steps. Hemolysin-mediated secretory expression; without tag removal step. Inclusion body formation of the tag-less IL6; in vitro refolding after solubilization; purification after refolding. Secretory expression into periplasmic space using a signal peptide. Inclusion body formation of the tag-less murine IL6; purification after solubilization. Inclusion body formation of the tag-less IL6; purification after in vitro refolding. Inclusion body formation of the tag-less IL6; in vitro refolding after solubilization; purification after refolding.
15 mg/l of mature IL6
Present work
50 mg/l of 6×His-tagged IL6
[16]
1621
transfected cells with the mature IL6 significantly increased the reporter activity in a sigmoidal manner, with a calculated EC50 of 117 ± 10 pM (n = 3). Our recombinant IL6 was even more effective than that prepared by other method (EC50 of ∼500 pM) in the similar receptor-activation assay [26]. Thus, we obtained fully active IL6 protein through E. coli overexpression using the novel NanoLucfusion approach. 3.4. Significance of the NanoLuc-fusion approach
2.6 mg/l of 6×His-tagged IL6
[17]
3 mg/l of 6×His-tagged IL6
[18]
Not doneb
[19]
18 g/l of hemolysin-tagged IL6 93.5 mg/l of mature IL6c
[20]
8–10 mg/l of mature IL6
[22]
5 mg/l of purified murine IL6d
[23]
25 mg/l of purified mature IL6
[24]
Not mentionede
[25]
[21]
In our previous [10] and present studies, the newly develop NanoLuc reporter [27] was used as a novel fusion partner for soluble overexpression of aggregation-prone proteins in E. coli. The NanoLuc fusion partner significantly improved the soluble overexpression of leukemia inhibitory factor (LIF) and IL6, which are liable to form inclusion bodies when overexpressed in E. coli because of their high aggregation propensity. As summarized in Table 1, the 6×His-tagged or the tag-less IL6 almost always formed inclusion bodies in E. coli, and the final yields of the folded IL6 varied from 3 mg/l to 90 mg/l, predominantly due to different in vitro refolding efficiencies that were difficult to control. The conventional GST, BMP and Trx fusion partners were not good enough for soluble overexpression of IL6 [17,19]. One report showed that NusA was efficient for soluble overexpression of IL6, but no purification and tag-removal processes were carried out [19], thus the real effect of NusA fusion partner for preparation of active IL6 protein was unknown. Our present NanoLuc fusion technology provided a good choice for quick preparation of fully active IL6 protein. In future studies, the NanoLuc fusion partner could also be applied to other aggregation-prone proteins for soluble overexpression in E. coli.
Acknowledgments We thank Promega Corporation for providing the plasmids encoding NanoLuc. This work was supported by the National Natural Science Foundation of China (31470767, 31270824) and the Fundamental Research Funds for the Central Universities (2000219098).
a
Amount of the final purified product from one liter of E. coli culture broth. 7.5 g/l of soluble NusA-fused IL6 was obtained as analyzed by SDS-PAGE under optimized fermentation conditions, but no purification steps were carried out. c 1.87 g of purified mature IL6 from 20 l of fermentation broth. d 25 mg of purified murine IL6 from 37 g wet cell pellet collected from 5 l of culture broth. e The final yield of the purified refolded IL6 was not mentioned; the IL6 inclusion body was ∼600 mg per liter of fermentation broth. b
(Fig. 2D), mature IL6 showed a typical ␣-helix-dominated spectrum, with two maximal negative peaks at 209 nm and 222 nm. The estimated ␣-helix content from the circular dichroism spectrum of the mature IL6 was ∼56%, which was consistent with the value calculated from the crystal structure of IL6 [14]. Thus, we deduced that the recombinant, mature IL6 formed a four-helix bundle structure with two correct disulfide bonds. From one liter of the E. coli culture broth, we could typically obtain approximately 15 mg of mature IL6 protein, which was much higher than previously reported yields of the mature IL6 [16–25]. Thus, the NanoLuc-fusion approach was efficient for overexpression of IL6 in E. coli. 3.3. Biological activity of the recombinant IL6 The biological activity of the mature IL6 was assayed as its receptor-activation potency using a STAT3-response NanoLuc reporter (Fig. 3). After the reporter was transfected into HEK293T cells that express the endogenous IL6 receptor, treatment of the
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[12] M. Pal, M.A. Febbraio, M. Whitham, From cytokine to myokine: the emerging role of interleukin-6 in metabolic regulation, Immunol. Cell Biol. 92 (2014) 331–339. [13] W. Hassan, L. Ding, R.Y. Gao, J. Liu, J. Shang, Interleukin-6 signal transduction and its role in hepatic lipid metabolic disorders, Cytokine 66 (2014) 133–142. [14] W. Somers, M. Stahl, J.S. Seehra, 1.9 A˚ crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling, EMBO J. 16 (1997) 989–997. [15] G. Skiniotis, M.J. Boulanger, K.C. Garcia, T. Walz, Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor, Nat. Struct. Mol. Biol. 12 (2005) 545–551. [16] N. Ahmed, A.U. Zafar, M.A. Khan, S. Tahir, M.I. Khan, H. Bashir, F. Khan, S. Sarwar, S. Ilyas, T. Husnain, Matrix-assisted refolding and purification of placenta-derived recombinant human interleukin-6 produced in Escherichia coli, Biotechnol. Appl. Biochem. 61 (2014) 541–548. [17] H. Nausch, J. Huckauf, R. Koslowski, U. Meyer, I. Broer, H. Mikschofsky, Recombinant production of human interleukin 6 in Escherichia coli, PLOS ONE 8 (2013) e54933. [18] V.A. Spiridonova, A.S. Lygina, M.M. Anohina, N.N. Tupitsyn, Preparation of functionally active recombinant human interleukin-6, Biochemistry (Mosc) 72 (2007) 424–429. [19] T.W. Kim, B.H. Chung, Y.K. Chang, Production of soluble human interleukin-6 in cytoplasm by fed-batch culture of recombinant E. coli, Biotechnol. Prog. 21 (2005) 524–531. [20] Y. Li, C.X. Chen, B.U. von Specht, H.P. Hahn, Cloning and hemolysin-mediated secretory expression of a codon-optimized synthetic human interleukin-6 gene in Escherichia coli, Protein Expr. Purif. 25 (2002) 437–447.
[21] D. Ejima, M. Watanabe, Y. Sato, M. Date, N. Yamada, Y. Takahara, High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli, Biotechnol. Bioeng. 62 (1999) 301–310. [22] I. Barthelemy, G. González de Buitrago, C. Carreiro, F. Roncal, A. Pérez-Aranda, G. Márquez, J.L. Barbero, Production and secretion of human interleukin 6 into the periplasm of Escherichia coli: efficient processing of N-terminal variants of hIL6 by the E. coli signal peptidase, J. Biotechnol. 27 (1993) 307–316. [23] J.G. Zhang, R.L. Moritz, G.E. Reid, L.D. Ward, R.J. Simpson, Purification and characterization of a recombinant murine interleukin-6. Isolation of N- and C-terminally truncated forms, Eur. J. Biochem. 207 (1992) 903–913. [24] R. Arcone, P. Pucci, F. Zappacosta, V. Fontaine, A. Malorni, G. Marino, G. Ciliberto, Single-step purification and structural characterization of human interleukin-6 produced in Escherichia coli from a T7 RNA polymerase expression vector, Eur. J. Biochem. 198 (1991) 541–547. [25] H. Yasueda, K. Nagase, A. Hosoda, Y. Akiyama, K. Yamada, High-level direct expression of semi-synthetic human interleukin-6 in Escherichia coli and production of N-terminus met-free product, Biotechnology (N.Y.) 8 (1990) 1036–1040. [26] http://www.promega.com/resources/protocols/product-information-sheets/ a/pgl4-47-vector-protocol [27] M.P. Hall, J. Unch, B.F. Binkowski, M.P. Valley, B.L. Butler, M.G. Wood, P. Otto, K. Zimmerman, G. Vidugiris, T. Machleidt, M.B. Robers, H.A. Benink, C.T. Eggers, M.R. Slater, P.L. Meisenheimer, D.H. Klaubert, F. Fan, L.P. Encell, K.V. Wood, Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate, ACS Chem. Biol. 7 (2012) 1848–1857.
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Efficient overexpression of human interleukin-6 in Escherichia coli using nanoluciferase as a fusion partner Ben-Jun Ji a,b , Ge Song b , Zhou Zhang a , Zhan-Yun Guo b,∗ a b
College of Life Sciences, Shanghai Normal University, Shanghai, China Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China
a r t i c l e
i n f o
Article history: Received 26 March 2015 Received in revised form 9 June 2015 Accepted 10 June 2015 Available online 14 June 2015 Keywords: Fusion partner E. coli NanoLuc Interleukin-6
a b s t r a c t Escherichia coli is widely used to produce recombinant proteins. For soluble overexpression, fusion technology has been developed using different fusion partners. In the present work, we used a newly developed nanoluciferase (NanoLuc) as a novel fusion partner for soluble overexpression of the aggregation-prone interleukin-6 (IL6). Soluble 6×His-NanoLuc-IL6 fusion protein was efficiently overexpressed in E. coli at a yield of approximately 60 mg per liter of culture broth after purification. The fusion partner was removed by enterokinase digestion and monomeric mature IL6 was obtained at a final yield of approximately 15 mg per liter of culture broth. The recombinant IL6 protein was fully active in the receptor activation assay, with a measured EC50 value of 117 ± 10 pM (n = 3). Thus, the novel NanoLucfusion approach was efficient for the soluble overexpression of active IL6 protein in E. coli and could be applied to other aggregation-prone proteins in future studies. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli is widely used to produce recombinant proteins in both the laboratory and industry [1–3]. In past decades, numerous heterologous proteins have been successfully overexpressed in E. coli. However, some proteins are prone to form insoluble inclusion bodies when overexpressed in E. coli and are hard to be renatured in vitro. For soluble overexpression of these aggregationprone proteins in E. coli, fusion technology has been developed, in which a highly soluble fusion partner protein [4–7], such as glutathione S-transferase (GST), maltose-binding protein (MBP), SUMO, NusA, thioredoxin (Trx), or ubiquitin is covalently fused to the target protein, typically at the N-terminus. Unfortunately, the solubilizing effect of these fusion partners varies significantly with different target proteins [8,9]. In a recent work, we found that fusion of a newly developed small nanoluciferase (NanoLuc, 19 kDa, 171 amino acids) significantly improved the soluble overexpression of the aggregation-prone human leukemia inhibitory factor (LIF) in E. coli [10], suggesting that NanoLuc might be a novel fusion partner. Interleukin-6 (IL6), also named interferon-ˇ2 (IFNB2), B-cell stimulatory factor 2 (BSF-2), hybridoma growth factor (HGF), and hepatocyte stimulating factor (HSF) in early references, is a well defined pro- and anti-inflammatory cytokine/myokine
that is involved in nearly all pathophysiological states by binding and activating a heterodimeric IL6R/gp130 receptor [11–13]. Mature human IL6 protein comprises 184 amino acids and forms a four-helix bundle structure with two disulfide bonds [14,15]. Overexpression of IL6 in E. coli has been attempted by several laboratories [16–25]. However, the yield of soluble active IL6 was low because of its high aggregation propensity, although different fusion partners were used. In the present work, we applied the novel NanoLuc-fusion approach to IL6 and showed it to be an efficient approach for overexpression of fully active IL6 in E. coli. 2. Materials and methods 2.1. DNA manipulation The codon-optimized nucleotide sequence of mature human IL6 was chemically synthesized and ligated into the previously generated pNLuc vector [10], after digestion with restriction enzymes EcoRI and HindIII, resulting in the expression construct pNLuc/IL6. The coding sequence of human IL6 was confirmed by DNA sequencing. 2.2. Overexpression and purification of 6×His-NanoLuc-IL6
∗ Corresponding author. Tel.: +86 21 65988634; fax: +86 21 65988403. E-mail address: [email protected] (Z.-Y. Guo). http://dx.doi.org/10.1016/j.procbio.2015.06.008 1359-5113/© 2015 Elsevier Ltd. All rights reserved.
The expression construct pNLuc/IL6 was transformed into E. coli strain BL21(DE3) and transformants were cultured in liquid LB medium to OD600 nm = 1.0 at 37 ◦ C. Thereafter, the inducer isopropyl
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Fig. 1. Overexpression and purification of 6×His-NanoLuc-IL6. (A) Flowchart of the purification and maturation process. (B) Induced overexpression analyzed by nonreducing SDS-PAGE. Lane (−), total cell lysate before IPTG induction (from 30 l of culture broth); lane (+), total cell lysate after IPTG induction (from 30 l of culture broth). (C) Solubility analyzed by non-reducing SDS-PAGE. Lane s, supernatant after sonication (1.0 l of sonication supernatant); lane p, pellet after sonication (from 1.0 l of sonication mixture). (D) Ni2+ column purification analyzed by non-reducing SDS-PAGE. Lane f, flowthrough fraction (1.0 l of flowthrough); lane w, fraction washed by 30 mM imidazole (1.0 l of wash fraction); lane e, fraction eluted by 250 mM imidazole (1.0 l of eluent). (E) Gel filtration purification analyzed by non-reducing SDS-PAGE. Lane p1, the first peak eluted from the gel filtration column (1.0 l of eluent); lane p2, the second peak eluted from the gel filtration column (1.0 l of eluent). The band corresponding to 6×His-NanoLuc-IL6 is indicated by an asterisk (*).
-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM and the cells were continuously cultured at 17 ◦ C for ∼13 h with gentle shaking (100 rpm). Subsequently, the E. coli cells were harvested by centrifugation (6000 × g, 5 min), resuspended in lysis buffer (20 mM phosphate buffer, pH 7.5, 0.5 M NaCl), and lysed by sonication. After centrifugation (12,000 × g, 30 min), the supernatant was applied to an immobilized metal ion affinity chromatography (Ni2+ column) and the bound 6×HisNanoLuc-IL6 protein was eluted by lysis buffer containing 250 mM imidazole. The 6×His-NanoLuc-IL6 fraction eluted from the Ni2+ column was subsequently loaded onto a gel filtration column (TSKgel G2000SWXL, 7.8 mm × 300 mm, Sigma Aldrich, St. Louis, MO, USA), eluted with 20 mM Tris–Cl buffer (pH 8.5), manually collected and analyzed by non-reducing SDS-PAGE.
2.3. Enterokinase cleavage of 6×His-NanoLuc-IL6 The 6×His-NanoLuc-IL6 fraction eluted from the gel filtration column was subjected to enterokinase digestion. CaCl2 was added to a final concentration of 2.0 mM and enterokinase (New England Biolabs, Ipswich, MA, USA) was added to a final concentration of ∼2 ng/ml. Digestion was carried out at 25 ◦ C overnight. Thereafter, the digestion mixture was loaded onto a DEAE ion-exchange column (TSKgel DEAE-5PW, 7.5 mm × 75 mm, from Sigma Aldrich). The fractions eluted by a gradient of sodium chloride (in 20 mM phosphate buffer, pH 7.5) were manually collected and analyzed by non-reducing SDS-PAGE.
2.4. Circular dichroism measurement The concentration of the purified mature IL6 was quantified by the 2,2 -bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as standard. Its final concentration was adjusted to 0.1 mg/ml for circular dichroism measurement, which was performed on a Jasco-715 spectrometer at room temperature. The spectrum was scanned from 180 to 250 nm using a quartz cuvette with 1.0 mm path length.
2.5. Biological activity assay of the recombinant IL6 The biological activity of the recombinant IL6 was assayed as its receptor activation potency using a STAT3-response sis-inducible element (SIE)-controlled NanoLuc as a reporter [10]. The reporter construct pNL1.2/SIE was transiently transfected into HEK293T cells. Next day, the transfected cells were typsinized, seeded into a 96-well plate, and continuously cultured to approximately 90% confluence. Thereafter, the medium was removed and the assay solution (serum-free DMEM medium plus 1% bovine serum albumin), containing different concentrations of mature IL6, was added (100 l/well) and the cells were continuously cultured at 37 ◦ C for 7 h. Subsequently, the assay solution was removed and the cells were lysed by adding lysis solution (100 l/well, from Promega, Madison, WI, USA). The cell lysate was then transferred to a white, opaque 96-well plate (50 l/well) and the bioluminescence was immediately measured on a SpectroMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA), after addition of the diluted substrate (50 l/well, from Promega). The measured data were expressed as mean ± SE and fitted to a sigmoidal curve using the SigmaPlot10.0 software. 3. Results and discussion 3.1. Overexpression and purification of 6×His-NanoLuc-IL6 in E. coli Overexpression of soluble IL6 in E. coli is difficult because of its high aggregation propensity [16–25]. To increase solubility of IL6 in E. coli, we tried the novel NanoLuc fusion partner. The mature human IL6 gene was chemically synthesized using E. coli-optimized codons and ligated into a pNLuc vector, which carries the coding sequence of 6×His-NanoLuc upstream of the IL6 gene. The condon usage of the synthetic human IL6 gene was manually harmonized to make it consistent with the statistic codon usage of E. coli (http://www.kazusa.or.jp/java/codon table java/). The nucleotide and amino acid sequence of 6×His-NanoLuc-IL6 fusion protein is shown in supplementary Fig. S1. The overexpression, purification
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and maturation process of 6×His-NanoLuc-IL6 is shown in Fig. 1A. After the expression construct pNLuc/IL6 was transformed into E. coli strain BL21(DE3), transformants were cultured in liquid LB medium and induced by IPTG at low temperature (17 ◦ C). After induction, a strong protein band (indicated by an asterisk) with a molecular weight of ∼40 kDa appeared on non-reducing SDSPAGE (Fig. 1B), suggesting that monomeric 6×His-NanoLuc-IL6 (theoretical molecular weight of 42.8 kDa) was efficiently overexpressed after IPTG induction. We wanted to detect whether the fused or mature IL6 formed oligomers through intermolecular disulfide crosslinking in E. coli and in the later purification processes, thus we used the non-reducing SDS-PAGE throughout the experimental procedures, except when stated otherwise. After the E. coli cells were lysed by sonication, the fusion protein was predominantly present in the supernatant, as analyzed by nonreducing SDS-PAGE (Fig. 1C), suggesting that the overexpressed 6×His-NanoLuc-IL6 was soluble. Thus, the NanoLuc fusion partner significantly increased solubility of the aggregation-prone IL6 protein in E. coli. The soluble 6×His-NanoLuc-IL6 was initially purified by an immobilized metal-ion affinity chromatography based on the N-terminal 6×His-tag. As analyzed by non-reducing SDS-PAGE (Fig. 1D), monomeric 6×His-NanoLuc-IL6 was a major band in the eluted fraction, although some minor bands were also present. After purified by Ni2+ column, typically ∼100 mg of monomeric 6×HisNanoLuc-IL6 could be obtained from one liter of E. coli culture broth as analyzed by non-reducing SDS-PAGE and densitometry using BSA as a standard. Thereafter, 6×His-NanoLuc-IL6 was further purified by gel filtration and two peaks were eluted. As analyzed by non-reducing SDS-PAGE (Fig. 1E), the first peak (p1) was almost homogenous monomeric 6×His-NanoLuc-IL6 and the second peak (p2) contained other smaller proteins. After two-step purification, approximately 60 mg of monomeric 6×His-NanoLuc-IL6 protein was typically obtained from one liter of E. coli culture broth as analyzed by non-reducing SDS-PAGE and densitometry. Thus, soluble, monomeric 6×His-NanoLuc-IL6 fusion protein could be efficiently overexpressed in E. coli using the novel NanoLuc-fusion approach. Supplementary Fig. S1 related to this article can be found, in the online version, at doi:10.1016/j.procbio.2015.06.008 3.2. Preparation and characterization of mature IL6 To obtain the mature IL6 protein, the purified 6×His-NanoLucIL6 was treated with enterokinase, which cleaves the fusion protein at the C-terminus of the DDDDK sequence (Fig. S1). As analyzed by non-reducing SDS-PAGE (Fig. 2A), after enterokinase digestion, the band corresponding to 6×His-NanoLuc-IL6 (42.8 kDa) disappeared and two smaller bands appeared, suggesting that enterokinase had cleaved the fusion protein efficiently. To separate the cleaved 6×His-NanoLuc fusion partner from the mature IL6, the digestion mixture was loaded onto a DEAE ion-exchange column, because the cleaved 6×His-NanoLuc and the mature IL6 have opposite charges. The positively charged mature IL6 would flow through the column, while the negatively charged 6×His-NanoLuc would be bound by the positively charged DEAE resin, and would only be eluted by a salt gradient. As analyzed by non-reducing SDS-PAGE (Fig. 2B), the cleaved 6×His-NanoLuc and the mature IL6 were well separated by the ion-exchange chromatography, because each fraction contained a single protein band. The mature IL6 (theoretical value of 21.2 kDa) had a slightly larger apparent molecular weight on the non-reducing SDS-PAGE than the cleaved 6×His-NanoLuc (theoretical value of 21.6 kDa). After treatment by the reducing reagent dithiothreitol (DTT), which breaks disulfide bonds, the mobility of the mature IL6 decreased slightly on SDS-PAGE (Fig. 2C), suggesting that the mature IL6 formed intramolecular disulfide bonds that rendered the polypeptide chain more compact and thus ran faster on SDS-PAGE. As analyzed by circular dichroism spectroscopy
Fig. 2. Maturation and characterization of the recombinant IL6. (A) Enterokinase digestion analyzed by non-reducing SDS-PAGE. Lane (−), fusion protein before enterokinase digestion; lane (+), fusion protein after enterokinase digestion. The band corresponding to 6×His-NanoLuc-IL6 is indicated by an asterisk (*); the band corresponding to mature IL6 is indicated by a double asterisk (**); the band corresponding to the cleaved 6×His-NanoLuc is indicated by a hash (#). (B) Ionexchange chromatography purification analyzed by non-reducing SDS-PAGE. Lane f, flowthrough fraction; lane e, fraction eluted by a gradient of sodium chloride. (C) SDS-PAGE analysis of the mature IL6 before (−) and after (+) DTT treatment. (D) Circular dichroism spectrum of the mature IL6.
Fig. 3. Biological activity assay of mature IL6 using a STAT3-response NanoLuc reporter. The measured bioluminescence data are expressed as mean ± SE (n = 3) and fitted to a sigmoidal curve using SigmaPlot 10.0 software.
B.-J. Ji et al. / Process Biochemistry 50 (2015) 1618–1622 Table 1 Summary of the recombinant expression of IL6 in E. coli. Approach
Final yielda
Reference
Soluble expression of 6×His-NanoLuc-tagged IL6; tag removed by enterokinase after purification. Inclusion body formation of 6×His-tagged IL6; in vitro on-column refolding after solubilization; without tag removal step. Soluble expression of 6×His-tagged IL6 through coexpression of chaperones; without purification and tag removal steps; Solubilizing effect of GST was not good. Inclusion body formation of 6×His-tagged IL6; in vitro refolding by dialysis; without tag removal step. NusA, BMP, Trx, ubiquitin tags were tested and NusA had the best solubilizing effect; without purification and tag removal steps. Hemolysin-mediated secretory expression; without tag removal step. Inclusion body formation of the tag-less IL6; in vitro refolding after solubilization; purification after refolding. Secretory expression into periplasmic space using a signal peptide. Inclusion body formation of the tag-less murine IL6; purification after solubilization. Inclusion body formation of the tag-less IL6; purification after in vitro refolding. Inclusion body formation of the tag-less IL6; in vitro refolding after solubilization; purification after refolding.
15 mg/l of mature IL6
Present work
50 mg/l of 6×His-tagged IL6
[16]
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transfected cells with the mature IL6 significantly increased the reporter activity in a sigmoidal manner, with a calculated EC50 of 117 ± 10 pM (n = 3). Our recombinant IL6 was even more effective than that prepared by other method (EC50 of ∼500 pM) in the similar receptor-activation assay [26]. Thus, we obtained fully active IL6 protein through E. coli overexpression using the novel NanoLucfusion approach. 3.4. Significance of the NanoLuc-fusion approach
2.6 mg/l of 6×His-tagged IL6
[17]
3 mg/l of 6×His-tagged IL6
[18]
Not doneb
[19]
18 g/l of hemolysin-tagged IL6 93.5 mg/l of mature IL6c
[20]
8–10 mg/l of mature IL6
[22]
5 mg/l of purified murine IL6d
[23]
25 mg/l of purified mature IL6
[24]
Not mentionede
[25]
[21]
In our previous [10] and present studies, the newly develop NanoLuc reporter [27] was used as a novel fusion partner for soluble overexpression of aggregation-prone proteins in E. coli. The NanoLuc fusion partner significantly improved the soluble overexpression of leukemia inhibitory factor (LIF) and IL6, which are liable to form inclusion bodies when overexpressed in E. coli because of their high aggregation propensity. As summarized in Table 1, the 6×His-tagged or the tag-less IL6 almost always formed inclusion bodies in E. coli, and the final yields of the folded IL6 varied from 3 mg/l to 90 mg/l, predominantly due to different in vitro refolding efficiencies that were difficult to control. The conventional GST, BMP and Trx fusion partners were not good enough for soluble overexpression of IL6 [17,19]. One report showed that NusA was efficient for soluble overexpression of IL6, but no purification and tag-removal processes were carried out [19], thus the real effect of NusA fusion partner for preparation of active IL6 protein was unknown. Our present NanoLuc fusion technology provided a good choice for quick preparation of fully active IL6 protein. In future studies, the NanoLuc fusion partner could also be applied to other aggregation-prone proteins for soluble overexpression in E. coli.
Acknowledgments We thank Promega Corporation for providing the plasmids encoding NanoLuc. This work was supported by the National Natural Science Foundation of China (31470767, 31270824) and the Fundamental Research Funds for the Central Universities (2000219098).
a
Amount of the final purified product from one liter of E. coli culture broth. 7.5 g/l of soluble NusA-fused IL6 was obtained as analyzed by SDS-PAGE under optimized fermentation conditions, but no purification steps were carried out. c 1.87 g of purified mature IL6 from 20 l of fermentation broth. d 25 mg of purified murine IL6 from 37 g wet cell pellet collected from 5 l of culture broth. e The final yield of the purified refolded IL6 was not mentioned; the IL6 inclusion body was ∼600 mg per liter of fermentation broth. b
(Fig. 2D), mature IL6 showed a typical ␣-helix-dominated spectrum, with two maximal negative peaks at 209 nm and 222 nm. The estimated ␣-helix content from the circular dichroism spectrum of the mature IL6 was ∼56%, which was consistent with the value calculated from the crystal structure of IL6 [14]. Thus, we deduced that the recombinant, mature IL6 formed a four-helix bundle structure with two correct disulfide bonds. From one liter of the E. coli culture broth, we could typically obtain approximately 15 mg of mature IL6 protein, which was much higher than previously reported yields of the mature IL6 [16–25]. Thus, the NanoLuc-fusion approach was efficient for overexpression of IL6 in E. coli. 3.3. Biological activity of the recombinant IL6 The biological activity of the mature IL6 was assayed as its receptor-activation potency using a STAT3-response NanoLuc reporter (Fig. 3). After the reporter was transfected into HEK293T cells that express the endogenous IL6 receptor, treatment of the
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