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Expression, purification, and characterization of recombinant human L-chain ferritin
Protein Expression and Purification 119 (2016) 63e68
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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Expression, purification, and characterization of recombinant human L-chain ferritin Wenyan Zou, Xiaoyu Liu, Xi Zhao, Jie Wang, Dianhua Chen, Jiahuang Li, Lina Ji*, Zichun Hua** State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210046, Jiangsu, PR China
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a b s t r a c t
Article history: Received 22 July 2015 Received in revised form 17 November 2015 Accepted 19 November 2015 Available online 24 November 2015
Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human Lchain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy. © 2015 Elsevier Inc. All rights reserved.
Keywords: Ferritin Solubility Expression Purification Characterization Protein cage
1. Introduction Isolated from horse spleen for the first time in 1937, ferritins are a family of proteins in charge of iron storage, iron release [1] and detoxification [2]. It is known that the 24 subunits of ferritins selfassemble into spherical cage architectures with protein shells and iron cores. Subunit analysis reveals that mammalian ferritins are composed of two different subunits known as H- and L-chains [3]. Each subunit forms a bundle of four long and a fifth short a-helix with a short non-helical extension [4,5]. Two types of subunits in different proportions compose a complex of 24 subunits in human tissues and organs [6,7]. In addition, the ratio of both H- and Lchains would vary depending on the pathological changes [7,8]. With an external size of 12e13 nm and an interior cavity of 7e8 nm in diameter [6,9], the hollow protein shell of ferritin is capable of
Abbreviations: CD, circular dichroism; DLS, dynamic light scattering; E. coli, Escherichia coli; HSF, horse spleen ferritin; IPTG, isopropyl-b-D-thiogalactopyranoside; PBS, phosphate buffered saline; rLF, recombinant human L-chain ferritin; SDSPAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TEM, transmission electron microscopy; TET, tetracyclines; TF, trigger factor. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L. Ji), [email protected] (Z. Hua). http://dx.doi.org/10.1016/j.pep.2015.11.018 1046-5928/© 2015 Elsevier Inc. All rights reserved.
hosting up to 4500 iron atoms [6,10]. Its inner cavity has been exploited as a reaction chamber for the template synthesis of nanoparticles with a well-defined size and a narrow size distribution [11,12]. A recombinant human H-chain ferritin-iron oxide nano-composite has been investigated as a magnetic resonance contrast agent [13]. Furthermore, ferritin nanocages have been used to transport drugs to different targets through either genetic modification or chemical conjugation [14e16]. In 2006, recombinant human L-chain ferritin (rLF) was revealed to be composed of 24 subunits, with each monomer composed of a 17-residue four-helix bundle, and a fifth helix linked at the C-terminal end [17]. Although H- and L-chains share 53% identical amino acid sequences and have similar three-dimensional structures [6,18], they perform different functions during the process of iron deposition [19]. H-chain contains a catalytic ferroxidase site that catalyzes the oxidation of Fe (II) to Fe (III) [9,20]. In contrast to Hchain, L-chain shows a capacity to induce iron mineralization with higher efficiency [21]. L-chain provides efficient nucleation sites for iron, and accelerates the development of the iron core [22e24]. The most recent study on the specific functions of L-chain showed that the electrons released during iron-oxidation were transported across the ferritin cages specifically through the L-chain and the inverted electron transport through the L-chain also accelerated the demineralization of ferritin [25]. Besides, a new ferritin
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receptor specific to L-chain was also identified as Scara5 that binds ferritin and then stimulates its endocytosis from the cell surface with consequent iron delivery [26,27]. Therefore, it is necessary to investigate the potential applications of H- and L-chains according to their distinct properties. However, the applications of L-chain are quite limited, partly because it forms inclusion bodies in Escherichia coli (E. coli) and its yield is reported to as low as 2e5 mg/L from bacterial culture [28]. In this study, we significantly improve the soluble yield of recombinant L-chain ferritin. Here we report a study to obtain rLF with high purity from E. coli. The yield of rLF is improved to 10 mg/L from bacterial culture with purity up to 96%. Then the rLF was characterized by circular dichroism (CD) spectrometry for its secondary structure. Moreover, the rLF cages were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Vectors,strains and chemicals Plasmid pET-28a, plasmid pG-Tf2 and host strains E. coli BL21 (DE3) were maintained in our laboratory. TRIzol reagent was from Invitrogen. PrimeScript RT Reagent Kit, phusion DNA polymerase, dNTP mix, restriction endonucleases and T4 DNA ligase were obtained from TaKaRa. Plasmid isolation kit was purchased from Shanghai Bocai Company. The isopropyl-b-D-thiogalactopyranoside (IPTG), tetracyclines (TET) and phosphotungstic were purchased from Sangon Biotech. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein standard was obtained from Thermo Scientific. Horse spleen ferritin (HSF) was obtained from SigmaeAldrich. BCA protein assay kit was purchased from CWBio. The Ni2þ-Sepharose 6 Fast Flow was obtained from GE Healthcare. 2.2. Construction of plasmid pET-28a-rLF Total RNA was extracted by TRIzol reagent from human lung adenocarcinoma A549 cells, and used for the reverse transcription reaction by PrimeScript RT Reagent Kit. Then the product of the reverse transcription reaction was used for amplification of rLF cDNA with primers designed based on its sequence in Genbank (M10119.1). For subsequent cloning into the bacterial expression plasmid pET-28a, the primers introduced restriction endonuclease cleavage sites for BamHI and XhoI, respectively. The constructed plasmid pET-28a-rLF was verified by DNA sequencing. 2.3. Expression of rLF alone The BL21 transformed with pET-28a-rLF was cultured in LB medium containing 50 mg/L kanamycin. The LB medium was shaken at 37 C until the OD600 reached 0.8, then IPTG was added to a final concentration of 1 mM to induce rLF expression. The BL21 cells were further cultured for 15 h at 20 C or 5 h at 37 C, and collected by centrifugation at 5000 rpm for 10 min at 4 C. 2.4. Co-expression of rLFand molecular chaperones The BL21 transformed with pET-28a-rLF (kanamycin-resistant) and pG-Tf2 (chloramphenicol-resistant) was cultured in LB medium containing kanamycin (50 mg/L) and chloramphenicol (34 mg/L) at 37 C, until the OD600 reached 0.8. To induce expression of molecular chaperones, TET was then added at a final concentration of 20 ng/L. After 30 min, 1 mM IPTG was added to trigger the expression of rLF. Then BL21 cells were further cultured for 15 h at 20 C or 5 h at 37 C, and collected by centrifugation.
2.5. Purification of rLF BL21 cells co-expressing rLF and molecular chaperones were harvested by centrifugation, and resuspended in 10 mM phosphate buffered saline (PBS, pH7.4) for sonication on ice. The cell lysate was centrifuged at 12,000 rpm for 15 min at 4 C, and the supernatant was collected. After the addition of imidazole to a final concentration of 10 mM, the supernatant was applied onto a 10-ml Ni2þSepharose 6 Fast Flow column pre-equilibrated with 10 mM PBS containing 10 mM imidazole. The column was then washed with 10 and 100 mM imidazole to remove nonspecifically bound proteins, and eluted with 250 and 500 mM imidazole. The eluted fraction of rLF protein was dialyzed overnight in 10 mM PBS to remove imidazole. The purity of rLF was analyzed by 12% SDS-PAGE. The final protein concentration was measured by BCA protein assay kit to calculate its yield from bacterial culture. 2.6. Analysis of purified protein The purified rLF was prepared as described above. The ferritin from horse spleen (HSF) was regarded as a reference. Far-UV CD spectra of rLF and the HSF were measured by a JASCO J-810 spectropolarimeter at 25 C. The protein samples were diluted to 5 mg/ mL, and transferred to a 0.05-cm path length quartz cuvette. For each spectrum, an average of three scans was obtained at a scan rate of 50 nm/min, and the data were presented as relative ellipticity (mdeg) in the range of 200e250 nm. Each TEM sample was prepared by depositing a drop of protein solution (1 mg/mL) on the surface of a copper net coated with carbon. After adsorption for 5 min, the samples were stained by 2% phosphotungstic acid for 1e2 min. Images were obtained using a JEOL TEM (JEM-2100, Japan) at 200 kV. Every protein sample was imaged for at least three times independently and each sample was observed in more than five regions to avoid experimental errors. The size of the protein nanoparticles (1 mg/mL) was measured using DLS (Malvern Zetasizer Nano S90; UK), and the mean value of triplicate measurements was adopted for analysis. 3. Results 3.1. Identification of the optimized conditions for rLF expression It is common that recombinant proteins form inclusion bodies when they are overexpressed in prokaryotic expression system. Optimization of temperature for protein expression is suggested to be a feasible way to improve the solubility of recombinant proteins [29]. Here the expression of rLF was tested at 20 C and 37 C, respectively. After sonication and centrifugation, BL21 cell lysate was separated as supernatant and pellet fractions. Then the levels of rLF in cell lysate, supernatant and pellet fractions were analyzed by SDS-PAGE. As shown in Fig. 1, majority of rLF was in pellet fractions at both 20 C (lane 3) and 37 C (lane 6). The amount of soluble rLF in supernatant was not changed significantly when the temperature was lowered from 37 C (lane 5) to 20 C (lane 2). Molecular chaperones are known to facilitate proteins folding [29,30]. Co-expression of molecular chaperones with recombinant proteins improves the solubility of recombinant proteins [31]. E. coli trigger factor (TF) is a new chaperone-like factor, which plays synergistic roles with molecular chaperones GroEL-GroES to facilitate protein folding and enhance production of active proteins. When induced by TET, plasmid pG-Tf2 can synchronously express molecular chaperones, including GroES (10 kDa), GroEL (60 kDa) and Tf (56 kDa) [32,33]. Therefore, pG-Tf2 was transformed into BL21 together with pET-28a-rLF to investigate whether the molecular chaperones can increase rLF expression in soluble form.
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solubility of rLF was only 26.1% in the absence of chaperones. Small amount of chaperones from leakage expression leads to a more than 2-fold increase in solubility of rLF. When the expression of chaperones was fully induced by TET, the solubility of rLF was increased to 74.8%. Owing to the leakage expression of chaperones, there was no significant difference between the solubility of rLF under TET-uninduced and TET-induced conditions. Moreover, as suggested by Fig. 2B and Fig. 3B, the solubility of rLF induced at 20 C was always higher than its equivalent at 37 C. 3.2. The purification and characterization of rLF
Fig. 1. The effect of temperature on the solubility of rLF was revealed by 12% SDS-PAGE gel. Lane M, Protein marker; Lane 1e3, cell lysate, supernatant and pellet fractions from BL21 expressing rLF at 20 C; Lane 4e6, cell lysate, supernatant and pellet fractions from BL21 expressing rLF at 37 C.
Compared with the result from Fig. 1, soluble rLF in supernatant was increased in the presence of molecular chaperones when induced at 37 C (Fig. 2A, lane 1 and lane 3). Molecular chaperones GroEL and Tf can be seen in the 12% SDS-PAGE gel, however, GroES was too small to be seen. In our study, the densitometric values of protein bands in SDS-PAGE gel were obtained by Quantity One software. To avoid the interference from overexpressed chaperones, the cytoplasmic solubility of rLF was denoted as the densitometric values of soluble rLF in supernatant fraction versus total (soluble plus insoluble) rLF in cell lysate. As indicated by Fig. 2B, the solubility of rLF at 37 C was only 21.4% in the absence of chaperones. However, chaperones significantly improved the expression of rLF in soluble form. Even before the addition of TET, there was a leakage expression of chaperones, which resulted in a 2-fold increase in the solubility of rLF. When chaperones were induced by TET for its high expression, the solubility of rLF was significantly improved to 50.3%. The effect of low temperature on expression of soluble rLF was also explored in the presence of molecular chaperones. As indicated by Fig. 3A (lane 1 and lane 3), the solubility of rLF was significantly improved in the presence of molecular chaperone at 20 C, compared with the result from Fig. 1. As shown by Fig. 3B, the
Based on its N-terminal His-tag from the backbone of pET-28a, the rLF was purified with Ni-affinity chromatography with a yield of 10 mg/L from bacterial culture. As shown in Fig. 4A, the purity of rLF was analyzed by 12% SDS-PAGE, and showed to be up to 96%. The CD spectrum of rLF was recorded to explore its secondary structure. As indicated in Fig. 4B, the CD spectrum of rLF showed to be slightly different from that of HSF. K2D3 software was used to estimate the secondary structure of proteins from CD spectra [34]. For HSF, the percentages of a-helix and b-strand were predicated to be 43.67% and 14.42%, respectively. Meanwhile, the percentages of a-helix and b-strand were 59.96% and 3.10% for rLF. TEM was used to observe the nanocages of rLF. As shown in Fig. 5A, rLF forms homogeneous nanoparticles with a narrow size distribution between 8 and 10 nm. TEM image in Fig. 5B indicates that HSF also assembles into 10e12 nm nanoparticles. The black dot in protein nanoparticles was speculated as the iron core of ferritin. Further evidence was provided by DLS for the size distribution of rLF nanocages. As indicated by Fig. 6A and B, the average diameters of rLF and HSF nanocages are 7.20 ± 0.83 nm and 11.44 ± 3.23 nm, respectively. The results from DLS are basically comparable to those from TEM. 4. Discussion Recombinant production of ferritin L-chain is more difficult than production of H-chain because L-chain is prone to form inclusion bodies when produced in prokaryotic expression system. A few years ago, Kim et al. developed a recombinant ferritin H/L-hybrid to enhance the cytoplasmic solubility of L-chain in E. coli [35]. In this study, the expression of rLF with an N-terminal His-tag was tested
Fig. 2. Effect of molecular chaperones on the solubility of rLF at 37 C. (A) The expression of rLF and molecular chaperones was revealed by 12% SDS-PAGE gel. IPTG and TET were used to induce expression of rFL and molecular chaperones, respectively. Lanes 1 and 2 showed protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG alone. Lanes 3 and 4 showed the protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG and 20 ng/mL TET. (B) The solubility of rLF at 37 C was quantified by densitometric analysis using Quantity One software. The data was analyzed by Student's two-tailed t test (n ¼ 3, bars represent means ±SD, * denotes p <0.05).
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Fig. 3. Effect of molecular chaperones on the solubility of rLF at 20 C. (A) The expression of rLF and molecular chaperones as revealed by 12% SDS-PAGE gel. IPTG and TET were used to induce expression of rFL and molecular chaperones, respectively. Lanes 1 and 2 showed protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG alone. Lanes 3 and 4 showed the protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG and 20 ng/mL TET. (B) The solubility of rLF at 20 C was quantified by densitometric analysis using Quantity One software. The data was analyzed by Student's two-tailed t test (n ¼ 3, bars represent means ±SD, * denotes p <0.05).
Fig. 4. Analysis of purified rLF by SDS-PAGE gel and CD spectrometry. (A) The purity of rLF was analyzed by 12% SDS-PAGE gel, and showed to be up to 96%. Lane M, Protein marker; Lane rLF, purified rLF. (B) The secondary structure of rLF and HSF was analyzed by CD spectrometry, and the data were presented as relative ellipticity (mdeg) in the range of 200e250 nm. The open circles denote rLF while the filled circles denote HSF.
at 20 C and 37 C. The result showed that lower temperature slightly increased the solubility of rLF from 21.4% (37 C) to 26.1% (20 C). Then the plasmid pG-Tf2 expressing a group of chaperones was introduced and showed a key role in improving the solubility of rLF. In the presence of chaperones at 20 C, the solubility of rLF was increased to 74.8%. In addition, we also found that leakage expression of molecular chaperones could lead to an increase in rLF expression in soluble form. Finally, under optimized conditions, we obtained rLF with a purity of 96%. The yield was 10 mg/L, which is much higher than previously reported 2e5 mg/L [28]. Human apoferritin and HSF share similar structures, and both are composed of H- and L-chains [36]. As indicated by CD spectra, the secondary structure of rLF and HSF are slightly different. Compared with HSF (90% L-chain and 10% H-chain), rLF is composed of a higher level of a-helix and lower level of b-strand, possibly due to their different protein sequences. The results from TEM and DLS showed that rLF was also able to self-assemble into spherical shells with a narrow size distribution, albeit smaller in size. Our present work suggests that co-expression of molecular
Fig. 5. TEM analysis of rLF and HSF. The nanocages of (A) rLF and (B) HSF were observed by TEM. Every protein sample was imaged for at least three times independently, and each sample was observed in more than five regions to avoid experimental error. The scale bar denotes 50 nm.
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Fig. 6. DLS analysis of rLF and HSF. The nanocage size distribution of (A) rLF and (B) HSF were determined by DLS. The mean value of triplicate measurements was adopted for analysis.
chaperones is critical for increasing the cytoplasmic solubility of rLF in bacteria. With the improved yield of rLF, we can further explore its biochemical and physiological properties, and investigate its potential role as a novel drug carrier in future studies. Acknowledgements The authors are grateful to grants from Ministry of Science and Technology (2012CB967004, 2014CB744501), the National Natural Science Foundation of China (31200583), the Jiangsu Provincial Nature Science Foundation (BE2013630), the Bureau of Science and Technology of Changzhou, Jiangsu, China (CZ20130011, CE20135013, CZ20120004, CM20122003 and WF201207).
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Expression, purification, and characterization of recombinant human L-chain ferritin Wenyan Zou, Xiaoyu Liu, Xi Zhao, Jie Wang, Dianhua Chen, Jiahuang Li, Lina Ji*, Zichun Hua** State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210046, Jiangsu, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 July 2015 Received in revised form 17 November 2015 Accepted 19 November 2015 Available online 24 November 2015
Ferritins form nanocage architectures and demonstrate their potential to serve as functional nanomaterials with potential applications in medical imaging and therapy. In our study, the cDNA of human Lchain ferritin was cloned into plasmid pET-28a for its overexpression in Escherichia coli. However, the recombinant human L-chain ferritin (rLF) was prone to form inclusion bodies. Molecular chaperones were co-expressed with rLF to facilitate its correct folding. Our results showed that the solubility of rLF was increased about 3-fold in the presence of molecular chaperones, including GroEL, GroES and trigger factor. Taking advantage of its N-terminal His-tag, rLF was then purified with Ni-affinity chromatography. With a yield of 10 mg/L from bacterial culture, the purified rLF was analyzed by circular dichroism spectrometry for its secondary structure. Furthermore, the rLF nanocages were characterized using dynamic light scattering and transmission electron microscopy. © 2015 Elsevier Inc. All rights reserved.
Keywords: Ferritin Solubility Expression Purification Characterization Protein cage
1. Introduction Isolated from horse spleen for the first time in 1937, ferritins are a family of proteins in charge of iron storage, iron release [1] and detoxification [2]. It is known that the 24 subunits of ferritins selfassemble into spherical cage architectures with protein shells and iron cores. Subunit analysis reveals that mammalian ferritins are composed of two different subunits known as H- and L-chains [3]. Each subunit forms a bundle of four long and a fifth short a-helix with a short non-helical extension [4,5]. Two types of subunits in different proportions compose a complex of 24 subunits in human tissues and organs [6,7]. In addition, the ratio of both H- and Lchains would vary depending on the pathological changes [7,8]. With an external size of 12e13 nm and an interior cavity of 7e8 nm in diameter [6,9], the hollow protein shell of ferritin is capable of
Abbreviations: CD, circular dichroism; DLS, dynamic light scattering; E. coli, Escherichia coli; HSF, horse spleen ferritin; IPTG, isopropyl-b-D-thiogalactopyranoside; PBS, phosphate buffered saline; rLF, recombinant human L-chain ferritin; SDSPAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TEM, transmission electron microscopy; TET, tetracyclines; TF, trigger factor. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L. Ji), [email protected] (Z. Hua). http://dx.doi.org/10.1016/j.pep.2015.11.018 1046-5928/© 2015 Elsevier Inc. All rights reserved.
hosting up to 4500 iron atoms [6,10]. Its inner cavity has been exploited as a reaction chamber for the template synthesis of nanoparticles with a well-defined size and a narrow size distribution [11,12]. A recombinant human H-chain ferritin-iron oxide nano-composite has been investigated as a magnetic resonance contrast agent [13]. Furthermore, ferritin nanocages have been used to transport drugs to different targets through either genetic modification or chemical conjugation [14e16]. In 2006, recombinant human L-chain ferritin (rLF) was revealed to be composed of 24 subunits, with each monomer composed of a 17-residue four-helix bundle, and a fifth helix linked at the C-terminal end [17]. Although H- and L-chains share 53% identical amino acid sequences and have similar three-dimensional structures [6,18], they perform different functions during the process of iron deposition [19]. H-chain contains a catalytic ferroxidase site that catalyzes the oxidation of Fe (II) to Fe (III) [9,20]. In contrast to Hchain, L-chain shows a capacity to induce iron mineralization with higher efficiency [21]. L-chain provides efficient nucleation sites for iron, and accelerates the development of the iron core [22e24]. The most recent study on the specific functions of L-chain showed that the electrons released during iron-oxidation were transported across the ferritin cages specifically through the L-chain and the inverted electron transport through the L-chain also accelerated the demineralization of ferritin [25]. Besides, a new ferritin
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receptor specific to L-chain was also identified as Scara5 that binds ferritin and then stimulates its endocytosis from the cell surface with consequent iron delivery [26,27]. Therefore, it is necessary to investigate the potential applications of H- and L-chains according to their distinct properties. However, the applications of L-chain are quite limited, partly because it forms inclusion bodies in Escherichia coli (E. coli) and its yield is reported to as low as 2e5 mg/L from bacterial culture [28]. In this study, we significantly improve the soluble yield of recombinant L-chain ferritin. Here we report a study to obtain rLF with high purity from E. coli. The yield of rLF is improved to 10 mg/L from bacterial culture with purity up to 96%. Then the rLF was characterized by circular dichroism (CD) spectrometry for its secondary structure. Moreover, the rLF cages were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). 2. Materials and methods 2.1. Vectors,strains and chemicals Plasmid pET-28a, plasmid pG-Tf2 and host strains E. coli BL21 (DE3) were maintained in our laboratory. TRIzol reagent was from Invitrogen. PrimeScript RT Reagent Kit, phusion DNA polymerase, dNTP mix, restriction endonucleases and T4 DNA ligase were obtained from TaKaRa. Plasmid isolation kit was purchased from Shanghai Bocai Company. The isopropyl-b-D-thiogalactopyranoside (IPTG), tetracyclines (TET) and phosphotungstic were purchased from Sangon Biotech. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein standard was obtained from Thermo Scientific. Horse spleen ferritin (HSF) was obtained from SigmaeAldrich. BCA protein assay kit was purchased from CWBio. The Ni2þ-Sepharose 6 Fast Flow was obtained from GE Healthcare. 2.2. Construction of plasmid pET-28a-rLF Total RNA was extracted by TRIzol reagent from human lung adenocarcinoma A549 cells, and used for the reverse transcription reaction by PrimeScript RT Reagent Kit. Then the product of the reverse transcription reaction was used for amplification of rLF cDNA with primers designed based on its sequence in Genbank (M10119.1). For subsequent cloning into the bacterial expression plasmid pET-28a, the primers introduced restriction endonuclease cleavage sites for BamHI and XhoI, respectively. The constructed plasmid pET-28a-rLF was verified by DNA sequencing. 2.3. Expression of rLF alone The BL21 transformed with pET-28a-rLF was cultured in LB medium containing 50 mg/L kanamycin. The LB medium was shaken at 37 C until the OD600 reached 0.8, then IPTG was added to a final concentration of 1 mM to induce rLF expression. The BL21 cells were further cultured for 15 h at 20 C or 5 h at 37 C, and collected by centrifugation at 5000 rpm for 10 min at 4 C. 2.4. Co-expression of rLFand molecular chaperones The BL21 transformed with pET-28a-rLF (kanamycin-resistant) and pG-Tf2 (chloramphenicol-resistant) was cultured in LB medium containing kanamycin (50 mg/L) and chloramphenicol (34 mg/L) at 37 C, until the OD600 reached 0.8. To induce expression of molecular chaperones, TET was then added at a final concentration of 20 ng/L. After 30 min, 1 mM IPTG was added to trigger the expression of rLF. Then BL21 cells were further cultured for 15 h at 20 C or 5 h at 37 C, and collected by centrifugation.
2.5. Purification of rLF BL21 cells co-expressing rLF and molecular chaperones were harvested by centrifugation, and resuspended in 10 mM phosphate buffered saline (PBS, pH7.4) for sonication on ice. The cell lysate was centrifuged at 12,000 rpm for 15 min at 4 C, and the supernatant was collected. After the addition of imidazole to a final concentration of 10 mM, the supernatant was applied onto a 10-ml Ni2þSepharose 6 Fast Flow column pre-equilibrated with 10 mM PBS containing 10 mM imidazole. The column was then washed with 10 and 100 mM imidazole to remove nonspecifically bound proteins, and eluted with 250 and 500 mM imidazole. The eluted fraction of rLF protein was dialyzed overnight in 10 mM PBS to remove imidazole. The purity of rLF was analyzed by 12% SDS-PAGE. The final protein concentration was measured by BCA protein assay kit to calculate its yield from bacterial culture. 2.6. Analysis of purified protein The purified rLF was prepared as described above. The ferritin from horse spleen (HSF) was regarded as a reference. Far-UV CD spectra of rLF and the HSF were measured by a JASCO J-810 spectropolarimeter at 25 C. The protein samples were diluted to 5 mg/ mL, and transferred to a 0.05-cm path length quartz cuvette. For each spectrum, an average of three scans was obtained at a scan rate of 50 nm/min, and the data were presented as relative ellipticity (mdeg) in the range of 200e250 nm. Each TEM sample was prepared by depositing a drop of protein solution (1 mg/mL) on the surface of a copper net coated with carbon. After adsorption for 5 min, the samples were stained by 2% phosphotungstic acid for 1e2 min. Images were obtained using a JEOL TEM (JEM-2100, Japan) at 200 kV. Every protein sample was imaged for at least three times independently and each sample was observed in more than five regions to avoid experimental errors. The size of the protein nanoparticles (1 mg/mL) was measured using DLS (Malvern Zetasizer Nano S90; UK), and the mean value of triplicate measurements was adopted for analysis. 3. Results 3.1. Identification of the optimized conditions for rLF expression It is common that recombinant proteins form inclusion bodies when they are overexpressed in prokaryotic expression system. Optimization of temperature for protein expression is suggested to be a feasible way to improve the solubility of recombinant proteins [29]. Here the expression of rLF was tested at 20 C and 37 C, respectively. After sonication and centrifugation, BL21 cell lysate was separated as supernatant and pellet fractions. Then the levels of rLF in cell lysate, supernatant and pellet fractions were analyzed by SDS-PAGE. As shown in Fig. 1, majority of rLF was in pellet fractions at both 20 C (lane 3) and 37 C (lane 6). The amount of soluble rLF in supernatant was not changed significantly when the temperature was lowered from 37 C (lane 5) to 20 C (lane 2). Molecular chaperones are known to facilitate proteins folding [29,30]. Co-expression of molecular chaperones with recombinant proteins improves the solubility of recombinant proteins [31]. E. coli trigger factor (TF) is a new chaperone-like factor, which plays synergistic roles with molecular chaperones GroEL-GroES to facilitate protein folding and enhance production of active proteins. When induced by TET, plasmid pG-Tf2 can synchronously express molecular chaperones, including GroES (10 kDa), GroEL (60 kDa) and Tf (56 kDa) [32,33]. Therefore, pG-Tf2 was transformed into BL21 together with pET-28a-rLF to investigate whether the molecular chaperones can increase rLF expression in soluble form.
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solubility of rLF was only 26.1% in the absence of chaperones. Small amount of chaperones from leakage expression leads to a more than 2-fold increase in solubility of rLF. When the expression of chaperones was fully induced by TET, the solubility of rLF was increased to 74.8%. Owing to the leakage expression of chaperones, there was no significant difference between the solubility of rLF under TET-uninduced and TET-induced conditions. Moreover, as suggested by Fig. 2B and Fig. 3B, the solubility of rLF induced at 20 C was always higher than its equivalent at 37 C. 3.2. The purification and characterization of rLF
Fig. 1. The effect of temperature on the solubility of rLF was revealed by 12% SDS-PAGE gel. Lane M, Protein marker; Lane 1e3, cell lysate, supernatant and pellet fractions from BL21 expressing rLF at 20 C; Lane 4e6, cell lysate, supernatant and pellet fractions from BL21 expressing rLF at 37 C.
Compared with the result from Fig. 1, soluble rLF in supernatant was increased in the presence of molecular chaperones when induced at 37 C (Fig. 2A, lane 1 and lane 3). Molecular chaperones GroEL and Tf can be seen in the 12% SDS-PAGE gel, however, GroES was too small to be seen. In our study, the densitometric values of protein bands in SDS-PAGE gel were obtained by Quantity One software. To avoid the interference from overexpressed chaperones, the cytoplasmic solubility of rLF was denoted as the densitometric values of soluble rLF in supernatant fraction versus total (soluble plus insoluble) rLF in cell lysate. As indicated by Fig. 2B, the solubility of rLF at 37 C was only 21.4% in the absence of chaperones. However, chaperones significantly improved the expression of rLF in soluble form. Even before the addition of TET, there was a leakage expression of chaperones, which resulted in a 2-fold increase in the solubility of rLF. When chaperones were induced by TET for its high expression, the solubility of rLF was significantly improved to 50.3%. The effect of low temperature on expression of soluble rLF was also explored in the presence of molecular chaperones. As indicated by Fig. 3A (lane 1 and lane 3), the solubility of rLF was significantly improved in the presence of molecular chaperone at 20 C, compared with the result from Fig. 1. As shown by Fig. 3B, the
Based on its N-terminal His-tag from the backbone of pET-28a, the rLF was purified with Ni-affinity chromatography with a yield of 10 mg/L from bacterial culture. As shown in Fig. 4A, the purity of rLF was analyzed by 12% SDS-PAGE, and showed to be up to 96%. The CD spectrum of rLF was recorded to explore its secondary structure. As indicated in Fig. 4B, the CD spectrum of rLF showed to be slightly different from that of HSF. K2D3 software was used to estimate the secondary structure of proteins from CD spectra [34]. For HSF, the percentages of a-helix and b-strand were predicated to be 43.67% and 14.42%, respectively. Meanwhile, the percentages of a-helix and b-strand were 59.96% and 3.10% for rLF. TEM was used to observe the nanocages of rLF. As shown in Fig. 5A, rLF forms homogeneous nanoparticles with a narrow size distribution between 8 and 10 nm. TEM image in Fig. 5B indicates that HSF also assembles into 10e12 nm nanoparticles. The black dot in protein nanoparticles was speculated as the iron core of ferritin. Further evidence was provided by DLS for the size distribution of rLF nanocages. As indicated by Fig. 6A and B, the average diameters of rLF and HSF nanocages are 7.20 ± 0.83 nm and 11.44 ± 3.23 nm, respectively. The results from DLS are basically comparable to those from TEM. 4. Discussion Recombinant production of ferritin L-chain is more difficult than production of H-chain because L-chain is prone to form inclusion bodies when produced in prokaryotic expression system. A few years ago, Kim et al. developed a recombinant ferritin H/L-hybrid to enhance the cytoplasmic solubility of L-chain in E. coli [35]. In this study, the expression of rLF with an N-terminal His-tag was tested
Fig. 2. Effect of molecular chaperones on the solubility of rLF at 37 C. (A) The expression of rLF and molecular chaperones was revealed by 12% SDS-PAGE gel. IPTG and TET were used to induce expression of rFL and molecular chaperones, respectively. Lanes 1 and 2 showed protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG alone. Lanes 3 and 4 showed the protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG and 20 ng/mL TET. (B) The solubility of rLF at 37 C was quantified by densitometric analysis using Quantity One software. The data was analyzed by Student's two-tailed t test (n ¼ 3, bars represent means ±SD, * denotes p <0.05).
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Fig. 3. Effect of molecular chaperones on the solubility of rLF at 20 C. (A) The expression of rLF and molecular chaperones as revealed by 12% SDS-PAGE gel. IPTG and TET were used to induce expression of rFL and molecular chaperones, respectively. Lanes 1 and 2 showed protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG alone. Lanes 3 and 4 showed the protein levels in the supernatant and pellet fractions from cells induced by 1 mM IPTG and 20 ng/mL TET. (B) The solubility of rLF at 20 C was quantified by densitometric analysis using Quantity One software. The data was analyzed by Student's two-tailed t test (n ¼ 3, bars represent means ±SD, * denotes p <0.05).
Fig. 4. Analysis of purified rLF by SDS-PAGE gel and CD spectrometry. (A) The purity of rLF was analyzed by 12% SDS-PAGE gel, and showed to be up to 96%. Lane M, Protein marker; Lane rLF, purified rLF. (B) The secondary structure of rLF and HSF was analyzed by CD spectrometry, and the data were presented as relative ellipticity (mdeg) in the range of 200e250 nm. The open circles denote rLF while the filled circles denote HSF.
at 20 C and 37 C. The result showed that lower temperature slightly increased the solubility of rLF from 21.4% (37 C) to 26.1% (20 C). Then the plasmid pG-Tf2 expressing a group of chaperones was introduced and showed a key role in improving the solubility of rLF. In the presence of chaperones at 20 C, the solubility of rLF was increased to 74.8%. In addition, we also found that leakage expression of molecular chaperones could lead to an increase in rLF expression in soluble form. Finally, under optimized conditions, we obtained rLF with a purity of 96%. The yield was 10 mg/L, which is much higher than previously reported 2e5 mg/L [28]. Human apoferritin and HSF share similar structures, and both are composed of H- and L-chains [36]. As indicated by CD spectra, the secondary structure of rLF and HSF are slightly different. Compared with HSF (90% L-chain and 10% H-chain), rLF is composed of a higher level of a-helix and lower level of b-strand, possibly due to their different protein sequences. The results from TEM and DLS showed that rLF was also able to self-assemble into spherical shells with a narrow size distribution, albeit smaller in size. Our present work suggests that co-expression of molecular
Fig. 5. TEM analysis of rLF and HSF. The nanocages of (A) rLF and (B) HSF were observed by TEM. Every protein sample was imaged for at least three times independently, and each sample was observed in more than five regions to avoid experimental error. The scale bar denotes 50 nm.
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Fig. 6. DLS analysis of rLF and HSF. The nanocage size distribution of (A) rLF and (B) HSF were determined by DLS. The mean value of triplicate measurements was adopted for analysis.
chaperones is critical for increasing the cytoplasmic solubility of rLF in bacteria. With the improved yield of rLF, we can further explore its biochemical and physiological properties, and investigate its potential role as a novel drug carrier in future studies. Acknowledgements The authors are grateful to grants from Ministry of Science and Technology (2012CB967004, 2014CB744501), the National Natural Science Foundation of China (31200583), the Jiangsu Provincial Nature Science Foundation (BE2013630), the Bureau of Science and Technology of Changzhou, Jiangsu, China (CZ20130011, CE20135013, CZ20120004, CM20122003 and WF201207).
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