Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor

Biochimie 106 (2014) 140e148

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Research paper

Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor Sheng-Xiang He a, b, 1, Ge Song a, 1, Jia-Ping Shi b, Yu-Qi Guo a, Zhan-Yun Guo a, * a b

Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China Shanghai Toneker Biotechnology Co. Ltd., Shanghai 200092, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 June 2014 Accepted 20 August 2014 Available online 30 August 2014

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6
His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd ¼ 1.4 ± 0.4 nM, n ¼ 3), the overexpressed LIFR/ gp130 (Kd ¼ 115 ± 8 pM, n ¼ 3), and the endogenously expressed LIFR/gp130 (Kd ¼ 33.1 ± 3.2 pM, n ¼ 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligandereceptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.
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Keywords: Nanoluciferase Protein fusion tag Leukemia inhibitory factor Bioluminescence Receptor-binding assays

1. Introduction Nanoluciferase (NanoLuc), developed by Promega in 2012, is the so far brightest bioluminescent reporter [1]. The new reporter produces a glow-type bioluminescence with a long half-life, using furimazine and molecular oxygen as substrates. In our previous work, active NanoLuc protein was efficiently overexpressed in Escherichia coli and chemically conjugated to a protein hormone, insulin-like peptide 3 (INSL3), for novel ultrasensitive bioluminescent receptor-binding assays [2]. In the present work, we attempted to develop NanoLuc as a novel quantitative protein fusion tag for

* Corresponding author. Institute of Protein Research, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China. Tel.: þ86 21 65988634; fax: þ86 21 65988403. E-mail address: [email protected] (Z.-Y. Guo). 1 These authors contributed equally to this work.

both efficient overexpression and various bioluminescent quantitative assays. Leukemia inhibitory factor (LIF) is an interleukin 6 family cytokine identified in the 1980s [3,4]. Its name was initially derived from its ability to prevent the continued growth of myeloid leukemic cells by inducing their terminal differentiation. LIF elicits pleiotropic effects on a diverse range of cells, such as embryonic stem cells, primordial germ cells, neurons, adipocytes, and osteoblasts [5e11]. The biological functions of LIF are mediated by a heterodimeric cell membrane receptor composed of a LIFR subunit (gp190) as well as a gp130 subunit that is the common b-chain of the interleukin-6 family cytokine receptors [12e14]. The heterodimeric LIFR/gp130 receptor binds LIF with high affinity (dissociation constant Kd in the 10e200 pM range), while the LIFR subunit itself can also bind LIF with low affinity (Kd in the 1e3 nM range). The heterodimeric LIFR/gp130 receptor also mediates signaling from cardiotrophin-1 and oncostatin M, whereas the heterotrimeric complex of ciliary neutrophic factor receptor (CNTFR)/

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S.-X. He et al. / Biochimie 106 (2014) 140e148

LIFR/gp130 mediates signaling from cardiotrophin-like cytokine and ciliary neutrophic factor [15e17]. LIF is synthesized in vivo as a precursor that contains an N-terminal signal peptide. The mature human LIF protein contains 180 amino acids and forms a four helix bundle structure with three conserved disulfide bonds [18,19]. The LIF residues involved in receptor-binding have been identified and various agonists and antagonists have been developed [20e22]. LIF promotes long-term maintenance of embryonic stem cells by suppressing spontaneous differentiation. Significant quantities of biologically active LIF protein are, therefore, required for stem cell research. Although several approaches have been used in previous work [23e28], the final yield of the biologically active, monomeric LIF is still quite low due to the hydrophobic nature and three disulfide bonds of the LIF protein. In the present work, we used a novel NanoLuc-fusion approach for both efficient overexpression in E. coli and ultrasensitive bioluminescent receptor-binding assays of the LIF protein. The present NanoLuc-fusion approach could be extended to other proteins for both sampled preparation and bioluminescent quantitative assays in future studies. 2. Materials and methods 2.1. Generation of a pNLuc vector A chemically synthesized DNA linker encoding cleavage sites of the protease factor X and enterokinase was ligated into a pET vector pretreated with the restriction enzymes NdeI and EcoRI, resulting in the construct pET/linker. The coding region of NanoLuc (lacking a stop codon) was PCR-amplified using pNL1.2 (Promega, Madison, WI, USA) as the template. The amplified DNA fragment was then treated with NdeI and ligated into the pET/linker construct pretreated with NdeI. The resultant correct pET/NLuc construct was identified by DNA sequencing. Thereafter, the first Met residue of NanoLuc in the pET/NLuc construct was mutated to Thr by sitedirected mutagenesis in order to eliminate one NdeI cleavage site in the construct. The resultant pET-derived expression vector was designated as pNLuc that encodes an N-terminal 6
His-NanoLuctag and an optional C-terminal 6
His-tag. The coding sequence (including a stop codon) of the mature human LIF was chemically synthesized using the E. coli-optimized codons. After cleavage with the restriction enzymes EcoRI and HindIII, the synthetic human LIF gene was inserted into the pNLuc vector pretreated with same restriction enzymes. The coding sequence of LIF was confirmed by DNA sequencing. 2.2. Recombinant expression and purification of the NanoLuc-fused LIF The pNLuc/LIF construct was transformed into the E. coli strain Rosetta-gami 2 (DE3). The transformed cells were cultured in liquid LuriaeBertani (LB) medium at 37  C to OD600 ¼ 1.0. Thereafter, the inducer isopropyl b-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM and the cells continuously cultured at 25  C overnight. The E. coli cells were harvested by centrifugation (5000 g, 5 min), resuspended in lysis solution (20 mM phosphate, pH 7.4, 0.5 M NaCl) and lysed by sonication. After centrifugation (10,000 g, 30 min), the supernatant was subjected to an immobilized metal ion affinity chromatography (Ni2þ column) and the fusion protein eluted by an imidazole gradient. The eluted 6
HisNanoLuc-LIF fraction was then loaded onto a gel filtration column (TSKgel G2000SWxL, 7.8 mm
300 mm, SigmaAldrich, St. Louis, MO, USA) and eluted by an aqueous solution containing 20 mM TriseCl (pH 8.5) and 50 mM NaCl. The eluted fractions were manually collected and analyzed by non-reducing SDS-PAGE.

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2.3. Enterokinase cleavage of the NanoLu-fused LIF CaCl2 and urea stock solutions were added to the above eluted 6
His-NanoLuc-LIF fraction (protein concentration approximately 1 mg/ml) at final concentrations of 2.0 mM and 1.6 M, respectively. Then, enterokinase (New England Biolabs, Ipswich, MA, USA) was added to a final concentration of 2 ng/ml and the cleavage was carried out at 25  C overnight. Thereafter, the digestion mixture was dialyzed against 20 mM phosphate buffer (pH 7.4) and loaded onto a DEAE ion-exchange column (TSKgel DEAE-5PW, 7.5 mm
75 mm, from SigmaAldrich). The flowthrough fraction and fractions eluted by a sodium chloride gradient were manually collected and analyzed by non-reducing SDS-PAGE. 2.4. Circular dichroism measurement The purified mature LIF protein was quantitated by the 2,20 bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as a standard [29]. The final concentration of the recombinant LIF protein was adjusted to 0.1 mg/ml in 20 mM phosphate buffer (pH 7.4) for circular dichroism measurement that was performed on a Jasco-715 spectrometer at room temperature. The spectrum was scanned from 190 to 250 nm using a quartz cuvette with a 0.1 cm path length. J-700 for Windows Secondary Structural Estimation software (version 1.10.00) was used to assess the secondary structure content from the circular dichroism spectrum. 2.5. Biological activity assays For the inhibition assay of murine leukemia M1 cell proliferation, M1 cells were first suspended in assay medium (RPMI1640 medium plus 10% fetal bovine serum, 1% BSA, 100 U/ml penicillin and 100 mg/ml streptomycin) at the cell density of 105 cells/ml and then seeded into a 96-well plate (100 ml/well). Thereafter, the serially diluted protein in the assay medium was added (100 ml/ well) and the resultant final concentrations of 6
His-NanoLuc-LIF or mature LIF were indicated in the figures. The cells were continuously cultured at 37  C for 3 days and then the cell numbers were measured using a WST-8 cell counting kit. The measured 450 nm absorbance data were expressed as mean ± SE (n ¼ 3) and fitted to a sigmoidal curve Y ¼ min þ (max  min)/(1 þ 10xlgEC50) using SigmaPlot 10.0 software. For the STAT3 signaling activation assay, a STAT3-response sis-inducible element (SIE)-controlled NanoLuc reporter was used. The SIE-controlled NanoLuc reporter was constructed by insertion of a synthetic SIE DNA sequence into the pNL1.2 vector (Promega) between the SacI and XhoI restriction enzyme sites, resulting in a pNL1.2/SIE construct. The pNL1.2/SIE construct was then transiently transfected into HEK293T cells. The following day, the transfected cells were trypsinized, seeded into a 96-well plate, and continuously cultured in complete medium (DMEM medium plus 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin) for one day to approximately 90% confluence. Thereafter, the culture medium was removed and the activation solution (serum-free DMEM medium plus 1% BSA) containing different concentrations of mature LIF or 6
His-NanoLucLIF was added (200 ml/well). After continuous culturing at 37  C for 3 h, the assay solution was removed and the cells were lysed in lysis solution (Promega, 100 ml/well). The cell lysate was then transferred to a white opaque 96-well plate (50 ml/well) and bioluminescence was measured on a SpectroMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA) after addition of the diluted furimazine substrate solution (50-fold diluted in phosphate-buffered saline, 50 ml/well). The measured bioluminescent data were expressed as mean ± SE (n ¼ 3) and fitted to

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sigmoidal curves Y ¼ min þ (max  min)/(1 þ 10lgEC50x) using SigmaPlot 10.0 software. 2.6. Bioluminescence measurement The purified 6
His-NanoLuc-LIF protein was quantitated by the BCA method. After sequential dilution with a 1% BSA solution (in phosphate-buffered saline), the diluted enzyme was added into a white, opaque 96-well plate (10 ml/well). After addition of the lysis solution (50 ml/well, Promega) and diluted furimazine substrate solution (40 ml/well, diluted in phosphate-buffered saline), bioluminescence was immediately measured on a SpectroMax M5 plate reader (Molecular Devices) using the luminescence mode.

washed once by centrifugation with cold serum-free medium (200 ml/well). Cell pellets were then resuspended in phosphatebuffered saline (100 ml/well) and transferred to a white opaque 96-well plate (50 ml/well). The cells were then lysed in lysis solution (25 ml/well, Promega). After addition of the diluted furimazine substrate solution (25 ml/well), bioluminescence was immediately measured on a SpectroMax M5 plate reader (Molecular Devices) using the luminescence mode. The measured binding data were expressed as mean ± SE (n ¼ 3) and fitted to a one-site receptorbinding model using SigmaPlot 10.0 software. The nonspecific binding data were obtained by competition with 30 nM of mature LIF. 3. Results and discussion

2.7. Binding assays with the overexpressed receptor The human LIFR gene was purchased from Yeli BioTech (Shanghai, China). Its coding region (including a stop codon) was PCR-amplified, digested by restriction enzymes NheI and AgeI, and ligated into the pcDNA6 vector, resulting in the construct pcDNA6/ LIFR. The full length coding region of LIFR was confirmed by DNA sequencing. The human gp130 gene expression construct pENTER/ gp130 was purchased from Vigene Biosciences (Rockville, MD, USA). HEK293T cells were transiently transfected with pcDNA6/ LIFR with or without cotransfection of pENTER/gp130 using the transfection reagent Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Next day, the transfected cells were trypsinized, seeded into a 96-well plate and continuously cultured for 24e48 h to approximately 90% confluence in a CO2 incubator at 37  C. Thereafter, culture medium was removed and binding solution (serum-free DMEM medium plus 1% BSA, 100 ml/well) was added. For saturation assay, the binding solution contained varied concentrations of 6
His-NanoLuc-LIF as indicated in the figures. For competition assay, the binding solution contained constant concentration of 6
His-NanoLuc-LIF and varied concentrations of mature LIF as indicated in the figures. After incubation at 20e21  C for 2 h, the assay solution was removed and the adherent cells were washed with cold serum-free DMEM medium twice (200 ml/well/time). Finally, the cells were lysed in lysis solution (Promega, 100 ml/well) and the cell lysate (50 ml/well) was transferred to a white opaque 96-well plate. After addition of the diluted furimazine substrate solution (50-fold diluted in phosphate-buffered saline, 50 ml/well), bioluminescence was immediately measured on a SpectroMax M5 plate reader (Molecular Devices) using the luminescence mode. The measured binding data were expressed as mean ± SE (n ¼ 3) and fitted to a one-site receptor-binding model using SigmaPlot 10.0 software. For total saturation binding, hyperbolic curve Y ¼ BmaxX/ (Kd þ X) þ NsX was used; for competition binding, sigmoidal curve Y ¼ min þ (max  min)/(1 þ 10xlgIC50) was used. 2.8. Binding assays with the endogenously expressed receptor Suspension murine leukemia M1 cells were collected by centrifugation. Adherent HEK293T cells, HepG2 cells and NIH-3T3 cells were treated with 1e5 mM of ethylenediaminetetraacetic acid in phosphate-buffered saline, washed with serum-free medium, and then collected by centrifugation. The cell pellets were resuspended in the assay solution (serum-free medium plus 1% BSA) and seeded into 96-well filtration plates (50 ml/well). Thereafter, 50 ml of serially diluted protein in the assay solution, containing either varied concentrations of 6
His-NanoLuc-LIF (for saturation assays) or a constant concentration of 6
His-NanoLucLIF and varied concentrations of mature LIF (for competition assays), were added. After incubation at 20e21  C for 1 h, the assay solution was removed by centrifugation (400 g, 1 min) and the cells

3.1. A novel NanoLuc-fusion approach for efficient overexpression and ultrasensitive bioluminescent quantitative assays In our previous work, active NanoLuc was efficiently overexpressed in E. coli and chemically conjugated to the protein hormone INSL3 for ultrasensitive bioluminescent receptor-binding assays [2]. In the present work, we attempted to use NanoLuc as a novel protein fusion tag for both efficient overexpression in E. coli and various ultrasensitive bioluminescent quantitative assays, such as receptor-binding assays and immunoassays. For convenient cloning and inducible expression of the NanoLuc-fused proteins in E. coli, we generated a pNLuc expression vector using a pET expression vector as the template (Fig. 1A). To facilitate purification, a 6
His-tag was present at the N-terminus of NanoLuc, allowing the fusion protein to be conveniently purified by immobilized metal ion affinity chromatography (Ni2þ column). The expression construct also provided an optional C-terminal 6
Histag: if the inserted gene contained a stop codon, the C-terminal 6
His-tag would be absent; otherwise, the C-terminal 6
His-tag would be present at the C-terminus of the target protein. In order to remove the 6
His-NanoLuc-tag from the fusion protein after purification, protease factor X and enterokinase cleavage sites were introduced into the C-terminus of the NanoLuc-tag. For convenient cloning of a foreign gene into this vector, a multiple cloning site was included downstream of the coding sequence of the NanoLuc-tag. 3.2. Recombinant expression and purification of the NanoLuc-fused LIF LIF is an important cytokine for stem cell research with a four helix bundle structure and three conserved disulfide bonds (Fig. 1B). To efficiently overexpress LIF in E. coli, we attempted the novel NanoLuc-fusion approach. A synthetic gene encoding mature human LIF was ligated into the pNLuc vector and the resultant pNLuc/LIF construct encoded a 6
His-NanoLuc-LIF fusion protein (378 amino acids, 41.6 kDa, Supplementary Fig. s1) without the optional C-terminal 6
His-tag due to presence of a stop codon at the end of the synthetic LIF gene. To improve the expression level, the synthetic LIF gene was codon optimized for E. coli expression. The fusion protein was recombinantly expressed in the E. coli strain Rosetta-gami 2 (DE3), which favors formation of disulfide bonds in the cytosol. The NanoLuc-tagged LIF protein was overexpressed in E. coli and downstream processed according to a procedure shown in Fig. 2A. After IPTG induction, a strong protein band (indicated by an asterisk) with a molecular weight approximately 40 kDa appeared as analyzed by SDS-PAGE (Fig. 2B). The apparent molecular weight of the inducibly expressed protein was consistent with the theoretical value (41.6 kDa) of the 6
His-NanoLuc-LIF fusion protein, thus we deduced that the NanoLuc-fused LIF was efficiently overexpressed in E. coli. After the E. coli cells were lysed by

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Fig. 1. (A) The nucleotide sequence and amino acid sequence of the expression frame of pNLuc vector. The cleavage sites of unique restriction enzymes at the multiple cloning site are labeled. The coding sequence of NanoLuc is shown in blue and partially omitted. The protease cleavage sites for removal of the 6
His-NanoLuc-tag are labeled. The C-terminal 6
His-tag can be used or not depending upon the stop codon of the inserted gene. (B) The reported crystal structure of LIF (PDB ID: 1LKI). The three conserved disulfide bonds are shown in red and labeled.

Fig. 2. Purification and characterization of the NanoLuc-fused LIF and the mature LIF. (A) Flowchart of the purification and maturation process. (B) Non-reducing SDS-PAGE analysis of 6
His-NanoLuc-LIF. Lane b, total cell lysate before IPTG induction (from 40 ml of culture broth); lane a, total cell lysate after IPTG induction (from 40 ml of culture broth); lane s, supernatant after sonication (1.5 ml of sonication supernatant); lane p, pellet after sonication (from 1.5 ml of sonication mixture); lane f, flowthrough from the Ni2þ column (1.5 ml of flowthrough); lane w, wash fraction by 30 mM imidazole (1.5 ml of wash fraction); lane e, eluted fraction by 250 mM imidazole (0.5 ml of eluent); lane M, protein marker. The asterisk (*) indicates the band of 6
His-NanoLuc-LIF that has a theoretical molecular weight of 41.6 kDa. After electrophoresis, the gel was stained by Coomassie brilliant blue R250. (C) Nonreducing SDS-PAGE analyses of the mature LIF. Lane b, monomeric 6
His-NanoLuc-LIF eluted from gel filtration (2.0 ml of eluent); lane a, after enterokinase digestion (2.0 ml of digestion mixture); lane f, flowthrough from the ion-exchange column (5.0 ml of flowthrough); lane e, eluent from the ion-exchange column (1.0 ml of eluent); lane (), mature LIF without DTT treatment; lane (þ), mature LIF treated with 20 mM DTT; lane M, protein marker. The asterisk (*) indicates the band of 6
His-NanoLuc-LIF that has a theoretical molecular weight of 41.6 kDa. The double asterisk (**) indicates the band of mature LIF that has a theoretical molecular weight of 20.0 kDa. The octothorpe (#) indicates the band of 6
His-NanoLuc-tag that has a theoretical molecular weight of 21.6 kDa. The arrowhead indicates the band of reduced LIF protein without disulfide bonds. After electrophoresis, the gel was stained by Coomassie brilliant blue R250. (D) Circular dichroism spectrum of the mature LIF.

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sonication, the majority of the fusion protein was present in the supernatant (Fig. 2B), suggesting soluble 6
His-NanoLuc-LIF protein was obtained using this approach. Soluble 6
His-NanoLuc-LIF was then purified from the cell lysate using a Ni2þ column (data not shown). SDS-PAGE analysis (Fig. 2B) indicated that monomeric 6
His-NanoLuc-LIF was a major component in the eluted fraction, although minor components were also present. Up to 80 mg of the soluble fusion protein could be obtained per liter of culture broth. In our previous work, we attempted to overexpress an N-terminally 6
His-tagged LIF (6
His-LIF) in E. coli, but it formed inclusion bodies and could not be refolded. Therefore, NanoLuc-tag improved solubility of the recombinant LIF protein. The eluted fraction from the Ni2þ column was further purified using a gel filtration column and homogenous monomeric 6
His-NanoLuc-LIF fusion protein was obtained (Fig. 2C). From 1 L of LB culture broth, approximately 20 mg of purified monomeric 6
His-NanoLuc-LIF protein could typically be obtained, indicating that the NanoLuc-fusion approach was effective for recombinant expression of LIF in E. coli. 3.3. Preparation of the mature LIF from the Nanoluc-fused precursor The purified monomeric 6
His-NanoLuc-LIF fusion protein was cleaved by enterokinase to remove the 6
His-NanoLuc-tag and obtain mature LIF protein. As shown in Fig. 2C, the fusion protein band (molecular weight of 41.6 kDa and indicated by an asterisk) disappeared and two smaller bands appeared after enterokinase treatment. We deduced that these new bands corresponded to the 6
His-NanoLuc-tag (molecular weight of 21.6 kDa and indicated by an octothorpe) and the mature LIF (molecular weight of 20.0 kDa and indicated by a double asterisk). After removal of urea from the digestion solution through dialysis, the digestion mixture was applied to a DEAE ion-exchange column. Non-reducing SDS-PAGE analysis (Fig. 2C) indicated that the mature LIF flowed through the ion-exchange column because it was positively charged and thus could not bind the positively charged DEAE resin. The cleaved 6
His-NanoLuc-tag was eluted by a sodium chloride gradient as it was negatively charged and thus bound the positively charged DEAE resin. To test whether the mature LIF protein formed disulfide bonds, it was treated with the reducing reagent dithiothreitol (DTT) that can break the disulfide bonds of LIF protein. As analyzed by SDS-PAGE (Fig. 2C), the mobility rate of the mature LIF was significantly decreased after DTT treatment, suggesting that DTT reduced disulfide bonds present in the mature LIF and thus rendered the polypeptide chain less compact. So, we deduced that our mature LIF protein contained disulfide bonds that are essential for its biological activities. To test whether the mature LIF formed a native conformation, its secondary structure was assessed by circular dichroism spectroscopy (Fig. 2D). The measured spectrum had two maximal negative peaks at 209 nm and 222 nm, suggesting a typical a-helix-dominated conformation. In contrast, the 6
His-NanoLuc-tag had a bsheet-dominated conformation as analyzed by circular dichroism (Fig. s2). The a-helix content of the mature LIF estimated from the circular dichroism spectrum was 57%, consistent with the value (approximately 60%) calculated from the crystal structure of LIF. Thus, we deduced that the mature LIF protein formed a native four helix bundle structure with three correct disulfide linkages. 3.4. Biological activities of the mature LIF and the NanoLuc-fused LIF To test the biological activities of the mature LIF and the NanoLuc-fused LIF, we first measured their inhibitory effects on the proliferation of murine leukemia M1 cells (Fig. 3A). After addition

Fig. 3. Biological activities of the mature LIF and the NanoLuc-fused LIF. (A) Murine leukemia M1 cell proliferation inhibition assay. M1 cells were seeded in a 96-well plate and cultured in medium containing different concentrations of LIF, 6
His-NanoLuc-LIF, or 6
His-NanoLuc for three days and then the cell numbers were measured using the WST-8 kit. The measured absorbance data at 450 nm were expressed as mean ± SE (n ¼ 3) and fitted to sigmoidal curve or linear curve using SigmaPlot 10.0. The inner panel shows the pictures of M1 cells after treated with 5.0 pM of mature LIF or 6
HisNanoLuc-LIF for six days. (B) STAT3 signaling activation assay using a SIE-controlled NanoLuc reporter. HEK293T cells transfected with a SIE-controlled NanoLuc reporter were seeded into a 96-well plate and treated with different concentrations of LIF or 6
His-NanoLuc-LIF for 3 h. Thereafter, the cells were lysed and the NanoLuc activity was measured. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3) and fitted to sigmoidal curves using SigmaPlot 10.0.

of the mature LIF or 6
His-NanoLuc-LIF, the proliferation of M1 cells was significantly inhibited and typical sigmoidal inhibition curves were obtained with calculated EC50 values of 2.0 ± 0.5 pM (n ¼ 3) for both proteins. The measured EC50 values were consistent with previously reported values using LIF protein prepared by other methods [26,29]. Thus, both the mature LIF and the NanoLucfused LIF retained high biological activity in this assay. In contrast, 6
His-NanoLuc had no effect on proliferation of M1 cells, suggesting that the inhibitory effects of 6
NanoLuc-LIF arose from the fused LIF protein. After the suspension M1 cells were treated with mature LIF or 6
NanoLuc-LIF, they became adherent due to induced differentiation (Fig. 3A, inner panel), suggesting that the inhibitory effects on proliferation were not caused by toxicity of these proteins. In addition, we measured their ability to activate the STAT3 signaling using a NanoLuc reporter whose expression was controlled by a STAT3-response element (Fig. 3B). After the

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NanoLuc reporter was transfected into HEK293T cells, treatment of these cells with mature LIF or 6
His-NanoLuc-LIF significantly increased NanoLuc reporter activity, suggesting that HEK293T cells expressed endogenous LIFR/gp130 receptor. Typical sigmoidal activation curves were obtained with calculated EC50 values of 6.6 ± 0.8 pM (n ¼ 3) for mature LIF and 19.1 ± 1.6 pM (n ¼ 3) for 6
His-NanoLuc-LIF. Thus, both the mature LIF and the NanoLucfused LIF had high biological activity in the STAT3 signaling activation assay. To confirm the measured bioluminescence was mainly from the STAT3-response NanoLuc reporter rather than from the added 6
His-NanoLuc-LIF fusion protein, 6
His-NanoLuc-LIF was added to non-transfected HEK293T cells and the measured bioluminescence was very low (less than 1% of the bioluminescence from the STAT3-response NanoLuc reporter), demonstrating that the NanoLuc-tag had almost no influence on the STAT3 activation assay. In summary, the biological activity assays demonstrated that both the mature LIF and the NanoLuc-fused LIF had full biological activities. 3.5. Binding of the NanoLuc-fused LIF with the overexpressed receptor The functional heterodimeric LIFR/gp130 receptor has high binding affinity with LIF protein, while LIFR itself retains low binding affinity with LIF protein. To measure the binding of LIF protein with its receptor, previous studies used radioactive 125Ilabeled LIF as a radioactive tracer [13,20,30,31]. However, the use of radioactive tracers has drawbacks, such as their short half-life and radioactive hazards. In the present work, we attempted to use the NanoLuc-fused LIF as a novel, non-radioactive tracer because the NanoLuc-tag has bright bioluminescence and is more sensitive than radioactive iodine-125 [2]. First, we tested whether NanoLuc was enzymatically active when its C-terminus was fused with LIF. The measured bioluminescence of 6
His-NanoLuc-LIF was similar to that of the recombinant 6
His-NanoLuc, approximately 1.5
105 for both proteins. Thus, the fused LIF had no detriment to NanoLuc activity. We then tested whether the NanoLuc-fused LIF retained binding potency with its receptor. First, we measured the binding potency of 6
His-NanoLuc-LIF with the overexpressed human LIFR (Fig. 4A). The saturation binding data of 6
His-NanoLuc-LIF with HEK293T cells overexpressing human LIFR were well fitted by a one-site receptor-binding model with a calculated Kd of 1.4 ± 0.4 nM (n ¼ 3). This result is consistent with the previously reported value measured using 125I-labeled LIF [13], suggesting that the NanoLuc-fused LIF retained full LIFR-binding potency despite the large NanoLuc molecule fused to its N-terminus. Binding of the recombinant 6
His-NanoLuc reporter with HEK293T cells overexpressing LIFR was very low and the data fit a linear model, suggesting the measured binding was predominantly nonspecific. The binding data of 6
His-NanoLuc-LIF with HEK293T cells transfected with empty pcDNA6 vector also fit a linear model, suggesting the measured data were predominantly due to nonspecific binding. Thus, the endogenous LIF receptor in HEK293T cells was too low to be detected at a high tracer concentration range. The NanoLuc reporter is a hydrophilic protein, resulting in low levels of nonspecific binding of 6
His-NanoLuc protein. In contrast, LIF is a hydrophobic protein, resulting in higher levels of nonspecific binding of 6
HisNanoLuc-LIF. As the NanoLuc-fused LIF retained high binding affinity with LIFR, we used it as a novel bioluminescent tracer in competition LIFR-binding assays (Fig. 4B). As the concentration of the mature LIF (competitor) was increased, the bound NanoLucfused LIF (tracer) to the overexpressed LIFR was decreased in a typical sigmoidal manner with a calculated IC50 value of 8.5 ± 1.3 nM (n ¼ 3) that was consistent with the previously reported value measured using 125I-labeled LIF [20].

Fig. 4. Binding of the NanoLuc-fused LIF with the overexpressed LIFR. (A) Saturation receptor-binding assays. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3). The binding data of 6
His-NanoLuc-LIF with HEK293T cells transiently overexpressing human LIFR were fitted to a one-site receptor-binding model Y ¼ BmaxX/(Kd þ X) þ NsX using SigmaPlot 10.0. The binding data of 6
His-NanoLuc-LIF with HEK293T cells and the binding data of NanoLuc with HEK293T cells overexpressing LIFR were fitted to linear curves using SigmaPlot 10.0. (B) Competition binding of mature LIF with overexpressed LIFR using 6
His-NanoLuc-LIF as a tracer. HEK293T cells transiently overexpressing human LIFR were used as a receptor source. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3) and fitted to a sigmoidal curve using SigmaPlot 10.0.

LIF binds the functional heterodimeric LIFR/gp130 receptor with high affinity. Thus, we also measured the binding potency of the NanoLuc-fused LIF with the overexpressed LIFR/gp130 receptor (Fig. 5A). The saturation binding data of 6
His-NanoLucLIF with HEK293T cell overexpressing LIFR/gp130 were well fitted by a one-site receptor-binding model at a low concentration range with a calculated Kd of 115 ± 8 pM (n ¼ 3) that was consistent with previously reported values measured using 125Ilabeled LIF [13]. This result suggested that the NanoLuc-fused LIF retained high binding affinity with the heterodimeric LIFR/gp130 receptor. The binding data of 6
His-NanoLuc-LIF with nontransfected HEK293T cells could also be roughly fitted by a onesite receptor-binding model at a low concentration range (Fig. 5A), suggesting that HEK293T cells express endogenous LIFR/ gp130 receptor. The binding curve of 6
His-NanoLuc-LIF with HEK293T cells overexpressing gp130 was similar to that of 6
HisNanoLuc-LIF with non-transfected HEK293T cells, suggesting

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3.6. Binding of the NanoLuc-fused LIF with the endogenously expressed receptor

Fig. 5. Binding of the NanoLuc-fused LIF with the overexpressed LIFR/gp130. (A) Saturation receptor-binding assays. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3) and fitted to a one-site receptor-binding model Y ¼ BmaxX/(Kd þ X) þ NsX using SigmaPlot 10.0. (B) Competition binding of mature LIF with overexpressed LIFR/gp130 using 6
His-NanoLuc-LIF as a tracer. HEK293T cells transiently overexpressing both human LIFR and human gp130 were used as a receptor source. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3) and fitted to a sigmoidal curve using SigmaPlot 10.0.

gp130 itself could not bind 6
His-NanoLuc-LIF at low tracer concentration. The measured binding data of 6
His-NanoLuc-LIF with HEK293T cells overexpressing both LIFR and gp130 were significantly higher than those of 6
His-NanoLuc-LIF with HEK293T cells overexpressing LIFR itself, suggesting that the measured binding was primarily contributed by the heterodimeric LIFR/gp130 receptor at a low tracer concentration range. We also measured competition binding of mature LIF with the heterodimeric LIFR/gp130 receptor using HEK293T cells overexpressing both LIFR and gp130 as the receptor source (Fig. 5B). When 100 pM of 6
His-NanoLuc-LIF was used, the measured competition binding was predominantly from LIFR/gp130 as the tracer could not efficiently bind LIFR at such low concentrations. The competition binding data of the mature LIF with LIFR/gp130 receptor were also fitted by a typical sigmoidal curve with a calculated IC50 value of 230 ± 30 pM (n ¼ 3). In summary, NanoLuc-fused LIF retained high binding affinity with the overexpressed LIFR and the overexpressed LIFR/gp130, thus representing a novel bioluminescent tracer to monitor ligandereceptor interactions.

Our above results demonstrated that the NanoLuc-fused LIF could efficiently bind with the overexpressed LIFR and LIFR/gp130. To test the sensitivity of the novel bioluminescent tracer, we carried out binding assays using cells expressing endogenous LIF receptor. As shown in Fig. 6A, 6
His-NanoLuc-LIF bound the murine leukemia M1 cells in a typical saturation manner, with a calculated Kd of 33.1 ± 3.2 pM (n ¼ 3), consistent with the values measured using 125 I-labeled LIF [30,31]. The binding data of 6
His-NanoLuc with M1 cells were very low and in a linear manner, suggesting that the 6
His-NanoLuc-tag had no specific binding with M1 cells. The calculated maximal binding capacity (Bmax) of 6
His-NanoLuc-LIF with 105 M1 cells was 8915 ± 225 counts, equal to ~350 receptors per M1 cell (specific activity of 6
His-NanoLuc-LIF was 1.5
105 counts/fmol). The present receptor density value determined using the bioluminescent tracer was consistent with previous values measured using 125I-labeled LIF [30,31]. The Scatchard plot of 6
His-NanoLuc-LIF binding with M1 cells was linear (Fig. 6B), suggesting that only the high affinity binding site was present on M1 cells, consistent with previous results [31]. As shown in Fig. 6C, competition binding of the mature LIF on M1 cells was in a typical sigmoidal manner when the NanoLuc-fused LIF was used as a bioluminescent tracer, with a calculated IC50 value of 15.8 ± 3.2 pM (n ¼ 3). Thus, the bioluminescent tracer was sensitive enough to monitor ligandereceptor interactions using the endogenously expressed LIFR/gp130. As shown in Fig. 6D, we also measured the expression levels of endogenous LIFR/gp130 on other cells. Calculated from the almost saturated specific binding values, the high affinity LIFR/gp130 on HEK293T cells was approximately 125 receptors per cell, confirming that HEK293T cells expressed endogenous functional LIF receptor. Human liver HepG2 cells also expressed endogenous LIFR/gp130 with a calculated receptor density of approximately 250 receptors per cell. The mouse fibroblast cell line HIH-3T3 expressed high levels of endogenous LIFR/ gp130, with a calculated receptor density of approximately 700 receptors per cell. The bioluminescent tracer could, therefore, quantify endogenous LIF receptor in various cell lines with a high degree of sensitivity. Assuming that the minimal bioluminescence that could be accurately measured for specific binding was 500 counts and the assay used 3
105 cells (nonspecific binding would be approximately 1000 counts when 80 pM of tracer was used), the detection limit of the bioluminescent tracer would be lower than 10 receptors per cell (specific activity of 6
His-NanoLuc-LIF was 1.5
105 counts/fmol when measured using a SpectroMax M5 plate reader). Thus, the NanoLuc-fused LIF could efficiently bind with the endogenously expressed receptor with an extremely low limit of detection, representing a novel ultrasensitive bioluminescent tracer to study ligandereceptor interactions. 3.7. Significance of the NanoLuc-fusion approach The present NanoLuc-fusion approach was efficient for overexpression of LIF in E. coli. Using this approach, up to 5 mg of purified, biologically active, monomeric LIF protein could be obtained from 1 L of the E. coli culture broth. NanoLuc has a small size (171 amino acids), high physical stability, and high expression level in E. coli. These characteristics make it a good fusion partner for overexpression of proteins in E. coli, especially proteins prone to aggregation. NanoLuc has the brightest bioluminescence reported to date. When measured using a SpectroMax M5 plate reader, an instrument that is widely available in laboratories, the specific activity of NanoLuc was approximately 1.5
105 counts/fmol, much higher than the radioactivity of iodine-125 (~4.8
103 dpm/fmol).

S.-X. He et al. / Biochimie 106 (2014) 140e148

147

Fig. 6. Binding of the NanoLuc-fused LIF with the endogenously expressed receptor. (A) Saturation binding with murine leukemia M1 cells. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3). The total binding data of 6
His-NanoLuc-LIF were fitted to Y ¼ BmaxX/(Kd þ X) þ NsX, and the specific binding data to Y ¼ BmaxX/(Kd þ X) using SigmaPlot 10.0. The nonspecific binding data of 6
His-NanoLuc-LIF and the total binding data of 6
His-NanoLuc were fitted to linear curves using SigmaPlot 10.0. (B) Scatchard plot of the specific binding data of 6
His-NanoLuc-LIF with M1 cells. (C) Competition binding of mature LIF with M1 cells using 6
His-NanoLuc-LIF as a tracer. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3) and fitted to a sigmoidal curve using SigmaPlot 10.0. (D) Binding of 6
His-NanoLuc-LIF with other cell lines. The measured bioluminescence data were expressed as mean ± SE (n ¼ 3). The total binding was measured at two tracer concentrations and the nonspecific binding was obtained by competition with 30 nM of mature LIF. The receptor density was calculated from the average specific binding at the two tracer concentrations.

The highest specific activity of 125I-labeled LIF is 3e7
104 cpm/ fmol (multiple site labeling) [30], which is still lower than the specific activity of the NanoLuc-fused LIF (1.5
105 counts/fmol). Additionally, nonspecific binding of the NanoLuc-fused LIF was

quite low due to the highly hydrophilic property of the NanoLuctag. Thus, the NanoLuc-fused LIF is an ultrasensitive probe suitable for various quantitative assays, such as bioluminescent receptor-binding assays and immunoassays. Other possible

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applications of this novel, ultrasensitive, and non-radioactive tracer could be developed in future studies, such as binding with tissue slice and cross-linking with LIF receptor. In summary, the present NanoLuc-fusion strategy not only provides an efficient approach for preparation of recombinant proteins but also provides ultrasensitive bioluminescent probes for various quantitative assays. The NanoLuc-fused ultrasensitive bioluminescent tracer can be easily prepared through recombinant expression in E. coli or other host cells and conveniently used over a long term without the radioactive hazards, thus the NanoLuc-fusion approach would be widely applied to other proteins in future studies. Conflict of interest None. Acknowledgments We thank Promega Corporation for providing the plasmids encoding NanoLuc. This work was supported by the National Basic Research Program of China (973 Program, No. 2010CB912604), the National Natural Science Foundation of China (31270824, 30970609), and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2014.08.012. References [1] 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) 1848e1857. [2] L. Zhang, G. Song, T. Xu, Q.P. Wu, X.X. Shao, Y.L. Liu, Z.G. Xu, Z.Y. Guo, A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase, Biochimie 95 (2013) 2454e2459. [3] D.P. Gearing, N.M. Gough, J.A. King, D.J. Hilton, N.A. Nicola, R.J. Simpson, E.C. Nice, A. Kelso, D. Metcalf, Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF), EMBO J. 6 (1987) 3995e4002. [4] N.M. Gough, D.P. Gearing, J.A. King, T.A. Willson, D.J. Hilton, N.A. Nicola, D. Metcalf, Molecular cloning and expression of the human homologue of the murine gene encoding myeloid leukaemia-inhibitory factor, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 2623e2627. [5] N.M. Gough, R.L. Williams, D.J. Hilton, S. Pease, T.A. Willson, J. Stahl, D.P. Gearing, N.A. Nicola, D. Metcalf, LIF: a molecule with divergent actions on myeloid leukaemic cells and embryonic stem cells, Reprod. Fertil. Dev. 1 (1989) 281e288. [6] R. Kurzrock, Z. Estrov, M. Wetzler, J.U. Gutterman, M. Talpaz, LIF: not just a leukemia inhibitory factor, Endocr. Rev. 12 (1991) 208e217. [7] D.J. Hilton, LIF: lots of interesting functions, Trends Biochem. Sci. 17 (1992) 72e76. [8] P.H. Patterson, Leukemia inhibitory factor, a cytokine at the interface between neurobiology and immunology, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 7833e7835. [9] D. Metcalf, The unsolved enigmas of leukemia inhibitory factor, Stem Cells 21 (2003) 5e14. [10] H. Hirai, P. Karian, N. Kikyo, Regulation of embryonic stem cell self-renewal and pluripotency by leukaemia inhibitory factor, Biochem. J. 438 (2011) 11e23.
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