- Email: [email protected]
Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca2+-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans
Protein Expression and Purification 84 (2012) 116–122
Contents lists available at SciVerse ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca2+-binding protein of the bc-crystallin superfamily from Methanosarcina acetivorans Venkatraman Ramanujam, Kandala V.R. Chary, Sri Rama Koti Ainavarapu ⇑ Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India
a r t i c l e
i n f o
Article history: Received 27 February 2012 and in revised form 21 April 2012 Available online 9 May 2012 Keywords: Crystallin Polyprotein Crystallin dimer Protein engineering Lens protein Force spectroscopy
a b s t r a c t bc-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two bstrand rich greek-key motifs. Recently, a structurally closest member to the family of lens bc-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as Mcrystallin. Unlike lens bc-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically (15N) labeled protein with high yield for NMR studies is reported. The uniformly 15 N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [15N–1H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer. Ó 2012 Elsevier Inc. All rights reserved.
Introduction The major components of vertebrate eye lens are a-, b- and c-crystallin proteins. a-Crystallins are members of heat-shock protein family. b- and c-Crystallins have been grouped together as bc-crystallin superfamily for their similar structural properties [1–3]. Although there is no known function for the bc-crystallins in the lens, it has been implicated that their function could be to maintain calcium homeostasis [4,5]. Their structural and calcium binding properties have been fairly understood. Their structures contain b-strand rich greek-key motifs. Recently, for the first time, a structurally closest member (M-crystallin) to the family of lens bc-crystallins from the archaea Methanosarcina acetivorans has been described [6]. This study suggested that the protein might be one amongst the primordial members of the group of proteins from which lens crystallins arose. This study also demonstrated that bc-crystallins are present in all three kingdoms of life, thus making it the most prevalent and widely distributed ⇑ Corresponding author. E-mail address: [email protected] (S.R.K. Ainavarapu). 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.04.024
calcium-binding superfamily in nature [6]. There is also very high sequence similarity between lens bc-crystallins and M-crystallin [7]. Further, both M-crystallin and bc-crystallins have been found to bind to calcium with similar affinity. Although, the exact function of M-crystallin is yet to be revealed, it has been speculated that it might be involved in calcium homeostasis, similar to that of vertebrate bc-crystallin. In this present project, we set out to synthesize a dimer of M-crystallin, in which two monomers are linked by a ten amino acid residue long coding sequence. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy [8–11]. In these studies, polyproteins are mechanically stretched, one at a time, to measure their mechanical properties. However, the linker sequence in these polyproteins was mostly two amino acid residues long to facilitate the interaction between individual proteins within the polyprotein. Such short linker is expected to have an effect on the structure and stability of the constituting protein units in the polyprotein. In these studies, it is generally assumed that the structural and functional properties of proteins thus spliced are unaffected in their
V. Ramanujam et al. / Protein Expression and Purification 84 (2012) 116–122
dimeric or polymeric forms. However, this assumption was never experimentally verified. In this backdrop, we have used a glycine-rich linker (GGGGRSGGGG) to attach the two monomeric M-crystallin units in a head-to-tail fashion. The residues Arg-Ser (RS) in the linker peptide arise due to the iterative cloning method used in constructing the dimeric M-crystallin gene. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer and thus avoid any possible interaction between the two linked monomers. We used HSQC spectral signatures seen from the dimer, to qualitatively characterize its structure and understand the domain–domain interactions, if any, between the two monomeric units and compared them with that of the un-tethered monomer. Materials and methods Materials All chemicals used for the protein expression and purification were of analytical grade. Restriction enzymes and DNA ligase were from Fermantas, Taq DNA polymerase was from Qiagen. DNA primers were synthesized by Sigma–Aldrich. Bacterial strain and plasmid Escherichia coli DH5a was used as a host strain for cloning, while BLR(DE3) was used as the host strain for protein expression. The pQE80L (Qiagen) vector was used for both cloning and expression system.
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100 lg/ml ampicillin and the culture was grown at 37 °C by shaking at 200 rpm until the OD600 reached 0.8. 10% inoculum was used from the starter culture to inoculate 1L LB medium containing 100 lg/ml ampicillin. The culture was induced at an OD600 of 0.8, with IPTG (isopropyl-b-D-thiogalactopyranoside) at a final concentration of 500 lM, and then further grown for another 8 h at 25 °C. The bacterial cells were harvested by centrifugation at 6000 rpm for 20 min at 4 °C, and then resuspended in 10 ml of lysis buffer (50 mM Tris–Cl, 100 mM KCl containing 1 mM PMSF, pH 7.5). The cells kept on ice, were then lysed for 40 min using sonicator (Branson sonifier 450) at 40% amplitude setting with 5 s on-pulses and 4 s off-pulses. Subsequently, the supernatant was collected after centrifugation at 17,000 rpm for 45 min at 4 °C. The supernatant containing the soluble protein was filtered through 0.45 lm filter. The buffer equilibrated Ni–NTA resin (Qiagen) is now incubated with the supernatant for 2 h at 4 °C with gentle rocking. The supernatant containing the beads were packed in 15 ml chromatography column (Bio-Rad, Hercules, CA). After collecting the flow-through, the beads were washed with five column volume of wash buffer (50 mM Tris–Cl, 100 mM KCl, pH 7.5) containing 5, 10, 20 and, 50 mM imidazole. The protein was eluted with 50 mM Tris–Cl, 100 mM KCl at pH 7.5 with 250 mM imidazole. The eluted protein was concentrated in 15 ml centrifugal filters (Millipore) with 10 kDa cut-off membrane. The concentrated protein was further purified using a Hiload 16/60 Superdex 75 preparation grade column using an AKTA chromatography system from GE. Gel filtration was performed in 10 mM Tris buffer pH 7.5 containing 50 mM KCl and 10 mM CaCl2. The protein was eluted between 62% and 70% of the column volume. The protein was desalted by centrifugal filtration using 10 kDa cut-off membrane, and stored at 20 °C after lyophilization.
Construction of pQE80L-M-crystallin dimer by iterative cloning Fluorescence spectra The M-crystallin monomer unit is sub-cloned after PCR amplification of M-crystallin cDNA using the primers designed as shown in Fig. 1. The 50 primer contains a BamHI restriction site and that permitted in-frame cloning of the monomer into the expression vector pQE80L. The 30 primer contained a linker sequence and BglII restriction site in-frame with M-crystallin domains followed by stop codon and KpnI site. PCR was performed according to the following thermocycle. Initial denaturation at 94 °C for 3 min; 30 cycles of denaturation at 94 °C for 30 s; annealing at 65 °C for 1 min; and extension at 72 °C for 1 min; followed by final extension at 72 °C for 10 min. The amplified PCR product was purified using PCR purification kit (Qiagen) to remove unused primers and dNTPs. The purified PCR product was restricted with BamHI and KpnI and ligated into pQE80L vector which is restricted with same restriction sites. The positively transformed colonies were subjected to another step of cloning by restricting with BglII and KpnI and ligation with another insert of M-crystallin gene restricted with BamHI and KpnI. BamHI and BglII having same cohesive ends permit directional cloning thereby giving rise to a M-crystallin dimer construct embedded in pQE80L vector (Fig. 1). At the ligation site, there will be a six base-pair sequence (AGATCC) that is linking the genes of M-crystallin and codes for Arg-Ser. This makes the resulting linker sequence between the two M-crystallin proteins in the dimer to be the ten amino acid long peptide, (Gly)4-Arg-Ser-(Gly)4.
Fluorescence spectra were acquired using a spex Fluoromax-3 spectrofluorometer. Spectra were acquired using 10 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2, with 1 cm path length cuvette. The protein was excited at 295 nm and the fluorescence emission was collected from 310 to 400 nm. The excitation and emission bandwidths were 2 nm. Each spectrum was collected with a 2 s integration time. Circular dichroism (CD) spectra The CD absorption spectra were recorded on a JASCO J-810 spectropolarimeter. A spectral range of 190–250 nm in far-UV region was used for probing the secondary structure of the protein, and a spectral range of 250–450 nm in the near-UV and visible region was used for probing the tertiary structure of the proteins. Far-UV CD spectra were collected at 10 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2 with 1 mm path length cuvette. Near-UV CD spectra were collected at 100 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2 using 1 cm path length cuvette. All CD spectra were acquired at a scan speed of 50 nm/min and a response time of 2 s. Preparation of
15
N-labeled M-crystallin dimer
Protein expression and purification BLR(DE3) cells were freshly transformed with the pQE80L-Mcrystallin dimer construct. Single colony was inoculated and grown over night in 100 ml Luria–Bertani (LB)1 media containing 1 Abbreviations used: LB, Luria–Bertani; IPTG, isopropyl-b-D-thiogalactopyranoside; CD, circular dichroism.
Uniformly 15N-labeled M-crystallin dimer was prepared by growing freshly transformed BLR(DE3) cells with the pQE80L-MCrystallin dimer construction. 1 L of M9 minimal medium containing 15NH4Cl is the sole source of nitrogen. The M9 medium contained the following: 0.05 mM CaCl2, 2.0 mM MgSO4, 0.4% glucose and 0.04% CAS amino acid. The other steps of overexpression and purification were the same as described above.
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Fig. 1. (A) Map of the plasmid constructed for the expression of M-crystallin dimer in E. coli BLR(DE3). A synthesized DNA segment containing the two identical genes of Mcrystallin linked by 10-amino acid residue coding sequence (GGGGRSGGGG) was inserted between BamHI and KpnI sites through gene fusion technique. (B) Sequence of primers used for fusing two genes with a linker sequence.
NMR sample preparation
NMR experiment
The sample for NMR spectroscopy was prepared dissolving 10 mg of 15N-labeled protein in 540 ll of 10 mM Tris buffer (pH 7.5) containing 50 mM KCl, and 10 mM CaCl2. The sample volume was made up to 600 ll, by adding 60 ll of 2H2O. The concentration of the protein was estimated to be 1 mM.
NMR experiments were recorded at 298 K on a Bruker Avance 800 MHz spectrometer equipped with a 5 mm triple-resonance cryogenic probe. Experiments recorded with uniformly 15N-labeled M-crystallin dimer included sensitivity-enhanced 2D [15N–1H]HSQC using water-flip-back for minimizing water saturation [12].
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Results and discussion PQE80L-M-crystallin dimer construct The gene of M-crystallin dimer was constructed through iterative cloning method. In this method, two M-crystallin genes were fused using sticky ends created by the digestion of engineered restriction enzyme sites at the termini. The resulting gene consisted of a two identical copies of the gene linked by a ten amino acid residues long coding linker and the size was confirmed by restriction digestion (Fig. 2). The detailed sequence of the dimer gene was further confirmed by DNA sequencing. The advantage of this method is that the primers themselves include the gene sequence coding for the linking peptide between the protein units in the dimer. In principle, it is possible to vary the linker in terms of its length and sequence. Moreover, this might be a better strategy not only in engineering polyproteins consisting of several proteins linked in tandem but minimize the interactions between them by reducing their proximity.
Protein expression and purification The protein was expressed in BLR(DE3). The initial growth phase (OD600 up to 0.8) was performed at 37 °C with the protein induction carried out at different temperatures. Finally, induction at 25 °C gave the highest yield of the soluble protein. Similarly, optimization of IPTG concentration was carried out under different concentrations. 500 lM concentration of IPTG gave the highest yield of protein, and no further increase in the yield of the protein was seen at higher IPTG concentrations (Fig. 3). The induced protein was present in the soluble fraction of the lysate. The supernatant was separated by centrifugation and mixed with the Ni–NTA beads for allowing the histidine tagged induced protein to bind with the beads. The non-target proteins were removed by washing the beads with wash buffer containing different concentrations of imidazole (5, 10, 20 and 50 mM). The eluted protein was 85% pure.
Fig. 3. SDS–PAGE analysis of M-crystallin shown on a 15% SDS gel: lane 1, molecular weight marker; lane 2, purified M-crystallin monomer; lane 3, purified M-crystallin dimer; lane 4, uninduced culture; lane 5, induced culture with 0.5 mM IPTG;
Gel filtration chromatography was then performed using Superdex G75 column (Fig. 4) after calibrating the column with different protein molecular weight standards (Sigma) thereby increasing the purity to P98% as seen in the SDS-PAGE (Fig. 3). The yield of protein was 30 mg/L. The yield of dimer is similar to that of monomer indicating the ten amino acid residues long linker does not affect the solubility of the dimer. This is important in the case of polyproteins used in single molecule force spectroscopy. It is quite often that the solubility of the polyprotein is much less than the solubility of the monomer and polyproteins often go into inclusion bodies after overexpression [13]. Fluorescence spectra Fluorescence spectra of M-crystallin dimer in its native and denatured state are shown in Fig. 5. They were acquired by exciting
Fig. 2. Gel electrophoresis analysis of M-crystallin gene products shown on a 1% agarose gel: lane 1, DNA ladder; lane 2, Double restriction digestion showing Mcrystallin monomer insert (300 bp) and pQE80L vector (4800 bp). Lane 3, Double restriction digestion showing M-crystallin dimer insert (600 bp) and pQE80L vector (4800 bp).
Fig. 4. Size exclusion chromatography results. M-crystallin dimer and monomer are eluted as single peaks (c) and (e), respectively. The molecular mass standards are also shown: (a) bovine serum albumin (66 kDa), (b) carbonic anhydrase (29 kDa), (d) cytochrome C from horse heart (12.4 kDa) and (f) aprotinin (6.5 kDa).
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Fig. 5. Steady state fluorescence spectra of M-crystallin dimer. (A) Steady State fluorescence spectrum of M-crystallin dimer in native condition. (B) Steady State fluorescence spectrum of M-crystallin dimer in 6 M GdmCl denaturing conditions.
Fig. 6. CD spectra of M-crystallin dimer. (A) Far-UV CD spectrum of M-crystallin dimer. (B) Near-UV CD spectrum of M-crystallin dimer.
the protein at 295 nm. The fluorescence spectrum was collected from 310 to 400 nm. The peak maximum was obtained at 332 and 350 nm for the native (Fig. 5A) and the denatured states (Fig. 5B), respectively. The maximum arises from the fluorescence of four Trp residues, two from each domain. Furthermore, the red shift in the fluorescence observed upon addition of 6 M guanidine hydrochloride as denaturant indicates that the Trp moiety gets solvent exposed, which was earlier in the core of protein. The fluorescence properties of the dimer are identical to that of the monomer [6].
indicating that the protein predominantly adopts b-sheet conformation, as in the case of monomer [6] and also as predicted by JPRED (Fig. 7A) [14]. Further, we have used a deconvolution method (K2D2) proposed by Perez-Iratxeta et al. [15] to quantify the secondary structural elements from the far-UV CD spectrum. This method gave 5% of a-helix, 43% b-sheet and 52% random coil. These fractions are comparable with secondary structural elements calculated from the NMR structure of M-crystallin monomer (36% b-sheet, and 64% of the random coil). Furthermore, the near-UV CD spectrum suggests that the aromatic amino acid residues are in asymmetric environments and the spectral features are very similar to that of the monomer as reported by Barnwal et al. [16].
Circular dichroic spectra M-crystallin dimer was characterized using far-UV and near-UV CD spectroscopy. Figs. 6A and 6B show far-and near-UV CD spectra of M-crystallin dimer, respectively. The far-UV CD spectrum shows minimum only at 214 nm without any minima at 208 or 222 nm,
NMR characterization The amino acid sequence of M-crystallin dimer is shown in Fig. 7A. The 15N-labeled protein sample is prepared by growing
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Fig. 7. (A) Sequence of M-crystallin dimer with the predicted secondary structure elements obtained by JPRED E: b-strand, H: a-helix (http://www.compbio.dundee.ac.uk/ ~www-jpred/). (B) Sensitivity enhanced 2D [15N–1H] HSQC of uniformly 15N-labeled M-crystallin monomer (in blue) and dimer (red). The spectrum was acquired with 2048 data points in t2 and 64 in t1 dimension with spectral widths of 11 and 28 ppm along 1H and 15N dimensions, respectively. The data were multiplied with shifted sine-square bell window function both along t1 and t2 axes, and zero-filled to 4096 along t2 dimension and to 256 along t1 dimension, prior to 2D Fourier transform.
the transformed BLR(DE3) cells containing pQE80L-M-crystallin dimer construct in a M9 minimal medium with 15NH4Cl as the sole source of nitrogen. The yield of protein is found to be 8 mg/L. The large spectral dispersion (3.5 ppm) of backbone amide 1H chemical shifts, as seen in the sensitivity-enhanced 2D [15N–1H]HSQC, indicates M-crystallin dimer is well folded as shown in Fig. 7B. A total of 98 correlation peaks seen in this HSQC indicates
that the dimeric protein shows up only one set of peaks as seen in the HSQC of M-crystallin monomer [16] in addition to correlation peaks coming from the linker sequence and N-terminal (histidine)6 tag. For comparison, HSQC of monomer protein is also shown as an overlay with that of the dimer. As is seen in the spectral overlay, the spectra of monomer and dimer look identical. This is another indication that the monomers in the dimeric protein have the same
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structure and that there is no interaction between the linker peptide and monomers. 1H, 13C and 15N resonance assignments of the dimer and the detailed structural characterization are underway. Conclusion In summary, we have synthesized a dimer of archaeal M-crystallin linked in tandem with a decapeptide and characterized its structure by optical and NMR spectroscopy. The structural characteristics of the dimer shown by fluorescence, circular dichroism and the HSQC spectral analysis are similar to that of M-crystallin monomer. This indicates that the linker peptide does not affect the structure of the protein. This methodology of linking proteins in tandem could improve the current strategy used in making polyproteins that are regularly used in single molecule force spectroscopy. As it would be impossible to do either NMR or crystallographic structural studies on large polyproteins containing many protein units, our methodology of making dimer to aid its structural characterization could bridge the structural characterization of monomers in bulk to the mechanical stability studies on polyproteins. Acknowledgments Authors would like to thank TIFR for financial assistance and the facilities provided by the National Facility for High Field NMR, supported by DST, DBT, CSIR, New Delhi and TIFR, Mumbai.
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Contents lists available at SciVerse ScienceDirect
Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep
Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca2+-binding protein of the bc-crystallin superfamily from Methanosarcina acetivorans Venkatraman Ramanujam, Kandala V.R. Chary, Sri Rama Koti Ainavarapu ⇑ Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India
a r t i c l e
i n f o
Article history: Received 27 February 2012 and in revised form 21 April 2012 Available online 9 May 2012 Keywords: Crystallin Polyprotein Crystallin dimer Protein engineering Lens protein Force spectroscopy
a b s t r a c t bc-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two bstrand rich greek-key motifs. Recently, a structurally closest member to the family of lens bc-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as Mcrystallin. Unlike lens bc-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically (15N) labeled protein with high yield for NMR studies is reported. The uniformly 15 N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [15N–1H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer. Ó 2012 Elsevier Inc. All rights reserved.
Introduction The major components of vertebrate eye lens are a-, b- and c-crystallin proteins. a-Crystallins are members of heat-shock protein family. b- and c-Crystallins have been grouped together as bc-crystallin superfamily for their similar structural properties [1–3]. Although there is no known function for the bc-crystallins in the lens, it has been implicated that their function could be to maintain calcium homeostasis [4,5]. Their structural and calcium binding properties have been fairly understood. Their structures contain b-strand rich greek-key motifs. Recently, for the first time, a structurally closest member (M-crystallin) to the family of lens bc-crystallins from the archaea Methanosarcina acetivorans has been described [6]. This study suggested that the protein might be one amongst the primordial members of the group of proteins from which lens crystallins arose. This study also demonstrated that bc-crystallins are present in all three kingdoms of life, thus making it the most prevalent and widely distributed ⇑ Corresponding author. E-mail address: [email protected] (S.R.K. Ainavarapu). 1046-5928/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pep.2012.04.024
calcium-binding superfamily in nature [6]. There is also very high sequence similarity between lens bc-crystallins and M-crystallin [7]. Further, both M-crystallin and bc-crystallins have been found to bind to calcium with similar affinity. Although, the exact function of M-crystallin is yet to be revealed, it has been speculated that it might be involved in calcium homeostasis, similar to that of vertebrate bc-crystallin. In this present project, we set out to synthesize a dimer of M-crystallin, in which two monomers are linked by a ten amino acid residue long coding sequence. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy [8–11]. In these studies, polyproteins are mechanically stretched, one at a time, to measure their mechanical properties. However, the linker sequence in these polyproteins was mostly two amino acid residues long to facilitate the interaction between individual proteins within the polyprotein. Such short linker is expected to have an effect on the structure and stability of the constituting protein units in the polyprotein. In these studies, it is generally assumed that the structural and functional properties of proteins thus spliced are unaffected in their
V. Ramanujam et al. / Protein Expression and Purification 84 (2012) 116–122
dimeric or polymeric forms. However, this assumption was never experimentally verified. In this backdrop, we have used a glycine-rich linker (GGGGRSGGGG) to attach the two monomeric M-crystallin units in a head-to-tail fashion. The residues Arg-Ser (RS) in the linker peptide arise due to the iterative cloning method used in constructing the dimeric M-crystallin gene. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer and thus avoid any possible interaction between the two linked monomers. We used HSQC spectral signatures seen from the dimer, to qualitatively characterize its structure and understand the domain–domain interactions, if any, between the two monomeric units and compared them with that of the un-tethered monomer. Materials and methods Materials All chemicals used for the protein expression and purification were of analytical grade. Restriction enzymes and DNA ligase were from Fermantas, Taq DNA polymerase was from Qiagen. DNA primers were synthesized by Sigma–Aldrich. Bacterial strain and plasmid Escherichia coli DH5a was used as a host strain for cloning, while BLR(DE3) was used as the host strain for protein expression. The pQE80L (Qiagen) vector was used for both cloning and expression system.
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100 lg/ml ampicillin and the culture was grown at 37 °C by shaking at 200 rpm until the OD600 reached 0.8. 10% inoculum was used from the starter culture to inoculate 1L LB medium containing 100 lg/ml ampicillin. The culture was induced at an OD600 of 0.8, with IPTG (isopropyl-b-D-thiogalactopyranoside) at a final concentration of 500 lM, and then further grown for another 8 h at 25 °C. The bacterial cells were harvested by centrifugation at 6000 rpm for 20 min at 4 °C, and then resuspended in 10 ml of lysis buffer (50 mM Tris–Cl, 100 mM KCl containing 1 mM PMSF, pH 7.5). The cells kept on ice, were then lysed for 40 min using sonicator (Branson sonifier 450) at 40% amplitude setting with 5 s on-pulses and 4 s off-pulses. Subsequently, the supernatant was collected after centrifugation at 17,000 rpm for 45 min at 4 °C. The supernatant containing the soluble protein was filtered through 0.45 lm filter. The buffer equilibrated Ni–NTA resin (Qiagen) is now incubated with the supernatant for 2 h at 4 °C with gentle rocking. The supernatant containing the beads were packed in 15 ml chromatography column (Bio-Rad, Hercules, CA). After collecting the flow-through, the beads were washed with five column volume of wash buffer (50 mM Tris–Cl, 100 mM KCl, pH 7.5) containing 5, 10, 20 and, 50 mM imidazole. The protein was eluted with 50 mM Tris–Cl, 100 mM KCl at pH 7.5 with 250 mM imidazole. The eluted protein was concentrated in 15 ml centrifugal filters (Millipore) with 10 kDa cut-off membrane. The concentrated protein was further purified using a Hiload 16/60 Superdex 75 preparation grade column using an AKTA chromatography system from GE. Gel filtration was performed in 10 mM Tris buffer pH 7.5 containing 50 mM KCl and 10 mM CaCl2. The protein was eluted between 62% and 70% of the column volume. The protein was desalted by centrifugal filtration using 10 kDa cut-off membrane, and stored at 20 °C after lyophilization.
Construction of pQE80L-M-crystallin dimer by iterative cloning Fluorescence spectra The M-crystallin monomer unit is sub-cloned after PCR amplification of M-crystallin cDNA using the primers designed as shown in Fig. 1. The 50 primer contains a BamHI restriction site and that permitted in-frame cloning of the monomer into the expression vector pQE80L. The 30 primer contained a linker sequence and BglII restriction site in-frame with M-crystallin domains followed by stop codon and KpnI site. PCR was performed according to the following thermocycle. Initial denaturation at 94 °C for 3 min; 30 cycles of denaturation at 94 °C for 30 s; annealing at 65 °C for 1 min; and extension at 72 °C for 1 min; followed by final extension at 72 °C for 10 min. The amplified PCR product was purified using PCR purification kit (Qiagen) to remove unused primers and dNTPs. The purified PCR product was restricted with BamHI and KpnI and ligated into pQE80L vector which is restricted with same restriction sites. The positively transformed colonies were subjected to another step of cloning by restricting with BglII and KpnI and ligation with another insert of M-crystallin gene restricted with BamHI and KpnI. BamHI and BglII having same cohesive ends permit directional cloning thereby giving rise to a M-crystallin dimer construct embedded in pQE80L vector (Fig. 1). At the ligation site, there will be a six base-pair sequence (AGATCC) that is linking the genes of M-crystallin and codes for Arg-Ser. This makes the resulting linker sequence between the two M-crystallin proteins in the dimer to be the ten amino acid long peptide, (Gly)4-Arg-Ser-(Gly)4.
Fluorescence spectra were acquired using a spex Fluoromax-3 spectrofluorometer. Spectra were acquired using 10 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2, with 1 cm path length cuvette. The protein was excited at 295 nm and the fluorescence emission was collected from 310 to 400 nm. The excitation and emission bandwidths were 2 nm. Each spectrum was collected with a 2 s integration time. Circular dichroism (CD) spectra The CD absorption spectra were recorded on a JASCO J-810 spectropolarimeter. A spectral range of 190–250 nm in far-UV region was used for probing the secondary structure of the protein, and a spectral range of 250–450 nm in the near-UV and visible region was used for probing the tertiary structure of the proteins. Far-UV CD spectra were collected at 10 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2 with 1 mm path length cuvette. Near-UV CD spectra were collected at 100 lM protein concentration in 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mM CaCl2 using 1 cm path length cuvette. All CD spectra were acquired at a scan speed of 50 nm/min and a response time of 2 s. Preparation of
15
N-labeled M-crystallin dimer
Protein expression and purification BLR(DE3) cells were freshly transformed with the pQE80L-Mcrystallin dimer construct. Single colony was inoculated and grown over night in 100 ml Luria–Bertani (LB)1 media containing 1 Abbreviations used: LB, Luria–Bertani; IPTG, isopropyl-b-D-thiogalactopyranoside; CD, circular dichroism.
Uniformly 15N-labeled M-crystallin dimer was prepared by growing freshly transformed BLR(DE3) cells with the pQE80L-MCrystallin dimer construction. 1 L of M9 minimal medium containing 15NH4Cl is the sole source of nitrogen. The M9 medium contained the following: 0.05 mM CaCl2, 2.0 mM MgSO4, 0.4% glucose and 0.04% CAS amino acid. The other steps of overexpression and purification were the same as described above.
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Fig. 1. (A) Map of the plasmid constructed for the expression of M-crystallin dimer in E. coli BLR(DE3). A synthesized DNA segment containing the two identical genes of Mcrystallin linked by 10-amino acid residue coding sequence (GGGGRSGGGG) was inserted between BamHI and KpnI sites through gene fusion technique. (B) Sequence of primers used for fusing two genes with a linker sequence.
NMR sample preparation
NMR experiment
The sample for NMR spectroscopy was prepared dissolving 10 mg of 15N-labeled protein in 540 ll of 10 mM Tris buffer (pH 7.5) containing 50 mM KCl, and 10 mM CaCl2. The sample volume was made up to 600 ll, by adding 60 ll of 2H2O. The concentration of the protein was estimated to be 1 mM.
NMR experiments were recorded at 298 K on a Bruker Avance 800 MHz spectrometer equipped with a 5 mm triple-resonance cryogenic probe. Experiments recorded with uniformly 15N-labeled M-crystallin dimer included sensitivity-enhanced 2D [15N–1H]HSQC using water-flip-back for minimizing water saturation [12].
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Results and discussion PQE80L-M-crystallin dimer construct The gene of M-crystallin dimer was constructed through iterative cloning method. In this method, two M-crystallin genes were fused using sticky ends created by the digestion of engineered restriction enzyme sites at the termini. The resulting gene consisted of a two identical copies of the gene linked by a ten amino acid residues long coding linker and the size was confirmed by restriction digestion (Fig. 2). The detailed sequence of the dimer gene was further confirmed by DNA sequencing. The advantage of this method is that the primers themselves include the gene sequence coding for the linking peptide between the protein units in the dimer. In principle, it is possible to vary the linker in terms of its length and sequence. Moreover, this might be a better strategy not only in engineering polyproteins consisting of several proteins linked in tandem but minimize the interactions between them by reducing their proximity.
Protein expression and purification The protein was expressed in BLR(DE3). The initial growth phase (OD600 up to 0.8) was performed at 37 °C with the protein induction carried out at different temperatures. Finally, induction at 25 °C gave the highest yield of the soluble protein. Similarly, optimization of IPTG concentration was carried out under different concentrations. 500 lM concentration of IPTG gave the highest yield of protein, and no further increase in the yield of the protein was seen at higher IPTG concentrations (Fig. 3). The induced protein was present in the soluble fraction of the lysate. The supernatant was separated by centrifugation and mixed with the Ni–NTA beads for allowing the histidine tagged induced protein to bind with the beads. The non-target proteins were removed by washing the beads with wash buffer containing different concentrations of imidazole (5, 10, 20 and 50 mM). The eluted protein was 85% pure.
Fig. 3. SDS–PAGE analysis of M-crystallin shown on a 15% SDS gel: lane 1, molecular weight marker; lane 2, purified M-crystallin monomer; lane 3, purified M-crystallin dimer; lane 4, uninduced culture; lane 5, induced culture with 0.5 mM IPTG;
Gel filtration chromatography was then performed using Superdex G75 column (Fig. 4) after calibrating the column with different protein molecular weight standards (Sigma) thereby increasing the purity to P98% as seen in the SDS-PAGE (Fig. 3). The yield of protein was 30 mg/L. The yield of dimer is similar to that of monomer indicating the ten amino acid residues long linker does not affect the solubility of the dimer. This is important in the case of polyproteins used in single molecule force spectroscopy. It is quite often that the solubility of the polyprotein is much less than the solubility of the monomer and polyproteins often go into inclusion bodies after overexpression [13]. Fluorescence spectra Fluorescence spectra of M-crystallin dimer in its native and denatured state are shown in Fig. 5. They were acquired by exciting
Fig. 2. Gel electrophoresis analysis of M-crystallin gene products shown on a 1% agarose gel: lane 1, DNA ladder; lane 2, Double restriction digestion showing Mcrystallin monomer insert (300 bp) and pQE80L vector (4800 bp). Lane 3, Double restriction digestion showing M-crystallin dimer insert (600 bp) and pQE80L vector (4800 bp).
Fig. 4. Size exclusion chromatography results. M-crystallin dimer and monomer are eluted as single peaks (c) and (e), respectively. The molecular mass standards are also shown: (a) bovine serum albumin (66 kDa), (b) carbonic anhydrase (29 kDa), (d) cytochrome C from horse heart (12.4 kDa) and (f) aprotinin (6.5 kDa).
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Fig. 5. Steady state fluorescence spectra of M-crystallin dimer. (A) Steady State fluorescence spectrum of M-crystallin dimer in native condition. (B) Steady State fluorescence spectrum of M-crystallin dimer in 6 M GdmCl denaturing conditions.
Fig. 6. CD spectra of M-crystallin dimer. (A) Far-UV CD spectrum of M-crystallin dimer. (B) Near-UV CD spectrum of M-crystallin dimer.
the protein at 295 nm. The fluorescence spectrum was collected from 310 to 400 nm. The peak maximum was obtained at 332 and 350 nm for the native (Fig. 5A) and the denatured states (Fig. 5B), respectively. The maximum arises from the fluorescence of four Trp residues, two from each domain. Furthermore, the red shift in the fluorescence observed upon addition of 6 M guanidine hydrochloride as denaturant indicates that the Trp moiety gets solvent exposed, which was earlier in the core of protein. The fluorescence properties of the dimer are identical to that of the monomer [6].
indicating that the protein predominantly adopts b-sheet conformation, as in the case of monomer [6] and also as predicted by JPRED (Fig. 7A) [14]. Further, we have used a deconvolution method (K2D2) proposed by Perez-Iratxeta et al. [15] to quantify the secondary structural elements from the far-UV CD spectrum. This method gave 5% of a-helix, 43% b-sheet and 52% random coil. These fractions are comparable with secondary structural elements calculated from the NMR structure of M-crystallin monomer (36% b-sheet, and 64% of the random coil). Furthermore, the near-UV CD spectrum suggests that the aromatic amino acid residues are in asymmetric environments and the spectral features are very similar to that of the monomer as reported by Barnwal et al. [16].
Circular dichroic spectra M-crystallin dimer was characterized using far-UV and near-UV CD spectroscopy. Figs. 6A and 6B show far-and near-UV CD spectra of M-crystallin dimer, respectively. The far-UV CD spectrum shows minimum only at 214 nm without any minima at 208 or 222 nm,
NMR characterization The amino acid sequence of M-crystallin dimer is shown in Fig. 7A. The 15N-labeled protein sample is prepared by growing
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Fig. 7. (A) Sequence of M-crystallin dimer with the predicted secondary structure elements obtained by JPRED E: b-strand, H: a-helix (http://www.compbio.dundee.ac.uk/ ~www-jpred/). (B) Sensitivity enhanced 2D [15N–1H] HSQC of uniformly 15N-labeled M-crystallin monomer (in blue) and dimer (red). The spectrum was acquired with 2048 data points in t2 and 64 in t1 dimension with spectral widths of 11 and 28 ppm along 1H and 15N dimensions, respectively. The data were multiplied with shifted sine-square bell window function both along t1 and t2 axes, and zero-filled to 4096 along t2 dimension and to 256 along t1 dimension, prior to 2D Fourier transform.
the transformed BLR(DE3) cells containing pQE80L-M-crystallin dimer construct in a M9 minimal medium with 15NH4Cl as the sole source of nitrogen. The yield of protein is found to be 8 mg/L. The large spectral dispersion (3.5 ppm) of backbone amide 1H chemical shifts, as seen in the sensitivity-enhanced 2D [15N–1H]HSQC, indicates M-crystallin dimer is well folded as shown in Fig. 7B. A total of 98 correlation peaks seen in this HSQC indicates
that the dimeric protein shows up only one set of peaks as seen in the HSQC of M-crystallin monomer [16] in addition to correlation peaks coming from the linker sequence and N-terminal (histidine)6 tag. For comparison, HSQC of monomer protein is also shown as an overlay with that of the dimer. As is seen in the spectral overlay, the spectra of monomer and dimer look identical. This is another indication that the monomers in the dimeric protein have the same
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structure and that there is no interaction between the linker peptide and monomers. 1H, 13C and 15N resonance assignments of the dimer and the detailed structural characterization are underway. Conclusion In summary, we have synthesized a dimer of archaeal M-crystallin linked in tandem with a decapeptide and characterized its structure by optical and NMR spectroscopy. The structural characteristics of the dimer shown by fluorescence, circular dichroism and the HSQC spectral analysis are similar to that of M-crystallin monomer. This indicates that the linker peptide does not affect the structure of the protein. This methodology of linking proteins in tandem could improve the current strategy used in making polyproteins that are regularly used in single molecule force spectroscopy. As it would be impossible to do either NMR or crystallographic structural studies on large polyproteins containing many protein units, our methodology of making dimer to aid its structural characterization could bridge the structural characterization of monomers in bulk to the mechanical stability studies on polyproteins. Acknowledgments Authors would like to thank TIFR for financial assistance and the facilities provided by the National Facility for High Field NMR, supported by DST, DBT, CSIR, New Delhi and TIFR, Mumbai.
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