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(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
Iterative cloning, overexpression, purification and isotopic labelingof an engineered dimer of a Ca2+-binding protein of the bc-crystallin superfamilyfrom 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 2012and in revised form 21 April 2012Available online 9 May 2012
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 b-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens bc-crystallinshas been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens bc-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimericgene 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 betweenthe individual crystallins in the dimer. This methodology would be highly beneficial in designing polypro-teins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly usedin single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coliBLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purifiedusing nickel affinity chromatography and then by size-exclusion chromatography. Further, a method topurify isotopically (15N) labeled protein with high yield for NMR studies is reported. The uniformly15N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced2D [15N–1H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in theHSQC, along with the structural characterization using optical spectroscopy, suggests that the domains inthe dimer possess similar structure as that of the monomer.
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Introduction
The major components of vertebrate eye lens are a-, b- andc-crystallin proteins. a-Crystallins are members of heat-shockprotein family. b- and c-Crystallins have been grouped togetheras bc-crystallin superfamily for their similar structural properties[1–3]. Although there is no known function for the bc-crystallinsin the lens, it has been implicated that their function could be tomaintain calcium homeostasis [4,5]. Their structural and calciumbinding properties have been fairly understood. Their structurescontain b-strand rich greek-key motifs. Recently, for the first time,a structurally closest member (M-crystallin) to the family of lensbc-crystallins from the archaea Methanosarcina acetivorans hasbeen described [6]. This study suggested that the protein mightbe one amongst the primordial members of the group of proteinsfrom which lens crystallins arose. This study also demonstratedthat bc-crystallins are present in all three kingdoms of life,thus making it the most prevalent and widely distributed
calcium-binding superfamily in nature [6]. There is also very highsequence similarity between lens bc-crystallins and M-crystallin[7]. Further, both M-crystallin and bc-crystallins have been foundto bind to calcium with similar affinity.
Although, the exact function of M-crystallin is yet to be re-vealed, it has been speculated that it might be involved in calciumhomeostasis, similar to that of vertebrate bc-crystallin. In thispresent project, we set out to synthesize a dimer of M-crystallin,in which two monomers are linked by a ten amino acid residuelong coding sequence. This methodology would be highly benefi-cial in designing polyproteins (two or more proteins linked in tan-dem to aid mechanical stretching studies) that are regularly usedin single-molecule force spectroscopy [8–11]. In these studies,polyproteins are mechanically stretched, one at a time, to measuretheir mechanical properties. However, the linker sequence in thesepolyproteins was mostly two amino acid residues long to facilitatethe interaction between individual proteins within the polypro-tein. Such short linker is expected to have an effect on the structureand stability of the constituting protein units in the polyprotein. Inthese studies, it is generally assumed that the structural and func-tional properties of proteins thus spliced are unaffected in their
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dimeric or polymeric forms. However, this assumption was neverexperimentally verified. In this backdrop, we have used a gly-cine-rich linker (GGGGRSGGGG) to attach the two monomericM-crystallin units in a head-to-tail fashion. The residues Arg-Ser(RS) in the linker peptide arise due to the iterative cloning methodused in constructing the dimeric M-crystallin gene. The linker se-quence in the target protein is long and flexible enough to reducethe proximity between the individual crystallins in the dimer andthus avoid any possible interaction between the two linked mono-mers. We used HSQC spectral signatures seen from the dimer, toqualitatively characterize its structure and understand the do-main–domain interactions, if any, between the two monomericunits and compared them with that of the un-tethered monomer.
Materials and methods
Materials
All chemicals used for the protein expression and purificationwere of analytical grade. Restriction enzymes and DNA ligase werefrom Fermantas, Taq DNA polymerase was from Qiagen. DNA prim-ers 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 expres-sion system.
Construction of pQE80L-M-crystallin dimer by iterative cloning
The M-crystallin monomer unit is sub-cloned after PCR amplifi-cation of M-crystallin cDNA using the primers designed as shownin Fig. 1. The 50 primer contains a BamHI restriction site and thatpermitted in-frame cloning of the monomer into the expressionvector pQE80L. The 30 primer contained a linker sequence and BglIIrestriction site in-frame with M-crystallin domains followed bystop codon and KpnI site. PCR was performed according to the fol-lowing thermocycle. Initial denaturation at 94 �C for 3 min; 30 cy-cles 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 at72 �C for 10 min. The amplified PCR product was purified usingPCR purification kit (Qiagen) to remove unused primers and dNTPs.The purified PCR product was restricted with BamHI and KpnI andligated into pQE80L vector which is restricted with same restric-tion sites. The positively transformed colonies were subjected toanother step of cloning by restricting with BglII and KpnI and liga-tion with another insert of M-crystallin gene restricted with BamHIand KpnI. BamHI and BglII having same cohesive ends permit direc-tional cloning thereby giving rise to a M-crystallin dimer constructembedded in pQE80L vector (Fig. 1). At the ligation site, there willbe a six base-pair sequence (AGATCC) that is linking the genes ofM-crystallin and codes for Arg-Ser. This makes the resulting linkersequence between the two M-crystallin proteins in the dimer to bethe ten amino acid long peptide, (Gly)4-Arg-Ser-(Gly)4.
Protein expression and purification
BLR(DE3) cells were freshly transformed with the pQE80L-M-crystallin dimer construct. Single colony was inoculated and grownover night in 100 ml Luria–Bertani (LB)1 media containing
100 lg/ml ampicillin and the culture was grown at 37 �C by shakingat 200 rpm until the OD600 reached 0.8. 10% inoculum was usedfrom the starter culture to inoculate 1L LB medium containing100 lg/ml ampicillin. The culture was induced at an OD600 of 0.8,with IPTG (isopropyl-b-D-thiogalactopyranoside) at a final concen-tration of 500 lM, and then further grown for another 8 h at 25 �C.The bacterial cells were harvested by centrifugation at 6000 rpmfor 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). Thecells kept on ice, were then lysed for 40 min using sonicator(Branson sonifier 450) at 40% amplitude setting with 5 s on-pulsesand 4 s off-pulses. Subsequently, the supernatant was collected aftercentrifugation at 17,000 rpm for 45 min at 4 �C. The supernatantcontaining the soluble protein was filtered through 0.45 lm filter.The buffer equilibrated Ni–NTA resin (Qiagen) is now incubated withthe supernatant for �2 h at 4 �C with gentle rocking. The superna-tant containing the beads were packed in 15 ml chromatography col-umn (Bio-Rad, Hercules, CA). After collecting the flow-through, thebeads 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 proteinwas concentrated in 15 ml centrifugal filters (Millipore) with10 kDa cut-off membrane. The concentrated protein was furtherpurified using a Hiload 16/60 Superdex 75 preparation grade columnusing an AKTA chromatography system from GE. Gel filtration wasperformed in 10 mM Tris buffer pH 7.5 containing 50 mM KCl and10 mM CaCl2. The protein was eluted between 62% and 70% of thecolumn volume. The protein was desalted by centrifugal filtrationusing 10 kDa cut-off membrane, and stored at �20 �C afterlyophilization.
Fluorescence spectra
Fluorescence spectra were acquired using a spex Fluoromax-3spectrofluorometer. Spectra were acquired using 10 lM proteinconcentration in 10 mM Tris buffer (pH 7.5) containing 50 mMKCl and 10 mM CaCl2, with 1 cm path length cuvette. The proteinwas excited at 295 nm and the fluorescence emission was collectedfrom 310 to 400 nm. The excitation and emission bandwidths were2 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-810spectropolarimeter. A spectral range of 190–250 nm in far-UV re-gion was used for probing the secondary structure of the protein,and a spectral range of 250–450 nm in the near-UV and visible re-gion was used for probing the tertiary structure of the proteins.Far-UV CD spectra were collected at 10 lM protein concentrationin 10 mM Tris buffer (pH 7.5) containing 50 mM KCl and 10 mMCaCl2 with 1 mm path length cuvette. Near-UV CD spectra werecollected at 100 lM protein concentration in 10 mM Tris buffer(pH 7.5) containing 50 mM KCl and 10 mM CaCl2 using 1 cm pathlength cuvette. All CD spectra were acquired at a scan speed of50 nm/min and a response time of 2 s.
Preparation of 15N-labeled M-crystallin dimer
Uniformly 15N-labeled M-crystallin dimer was prepared bygrowing freshly transformed BLR(DE3) cells with the pQE80L-M-Crystallin dimer construction. 1 L of M9 minimal medium contain-ing 15NH4Cl is the sole source of nitrogen. The M9 mediumcontained the following: 0.05 mM CaCl2, 2.0 mM MgSO4, 0.4% glu-cose and 0.04% CAS amino acid. The other steps of overexpressionand purification were the same as described above.
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NMR sample preparation
The sample for NMR spectroscopy was prepared dissolving10 mg of 15N-labeled protein in 540 ll of 10 mM Tris buffer (pH7.5) containing 50 mM KCl, and 10 mM CaCl2. The sample volumewas made up to 600 ll, by adding 60 ll of 2H2O. The concentrationof the protein was estimated to be 1 mM.
NMR experiment
NMR experiments were recorded at 298 K on a Bruker Avance800 MHz spectrometer equipped with a 5 mm triple-resonancecryogenic probe. Experiments recorded with uniformly 15N-labeledM-crystallin dimer included sensitivity-enhanced 2D [15N–1H]-HSQC using water-flip-back for minimizing water saturation [12].
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 M-crystallin linked by 10-amino acid residue coding sequence (GGGGRSGGGG) was inserted between BamHI and KpnI sites through gene fusion technique. (B) Sequence ofprimers used for fusing two genes with a linker sequence.
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Results and discussion
PQE80L-M-crystallin dimer construct
The gene of M-crystallin dimer was constructed through itera-tive cloning method. In this method, two M-crystallin genes werefused using sticky ends created by the digestion of engineeredrestriction enzyme sites at the termini. The resulting gene con-sisted of a two identical copies of the gene linked by a ten aminoacid residues long coding linker and the size was confirmed byrestriction digestion (Fig. 2). The detailed sequence of the dimergene was further confirmed by DNA sequencing. The advantageof this method is that the primers themselves include the gene se-quence coding for the linking peptide between the protein units inthe dimer. In principle, it is possible to vary the linker in terms ofits length and sequence. Moreover, this might be a better strategynot only in engineering polyproteins consisting of several proteinslinked in tandem but minimize the interactions between them byreducing 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 induc-tion carried out at different temperatures. Finally, induction at25 �C gave the highest yield of the soluble protein. Similarly, opti-mization of IPTG concentration was carried out under differentconcentrations. 500 lM concentration of IPTG gave the highestyield of protein, and no further increase in the yield of the proteinwas seen at higher IPTG concentrations (Fig. 3). The induced pro-tein was present in the soluble fraction of the lysate. The superna-tant was separated by centrifugation and mixed with the Ni–NTAbeads for allowing the histidine tagged induced protein to bindwith the beads. The non-target proteins were removed by washingthe beads with wash buffer containing different concentrations ofimidazole (5, 10, 20 and 50 mM). The eluted protein was 85% pure.
Gel filtration chromatography was then performed using SuperdexG75 column (Fig. 4) after calibrating the column with different pro-tein molecular weight standards (Sigma) thereby increasing thepurity to P98% as seen in the SDS-PAGE (Fig. 3). The yield of pro-tein was 30 mg/L. The yield of dimer is similar to that of monomerindicating the ten amino acid residues long linker does not affectthe solubility of the dimer. This is important in the case of polypro-teins used in single molecule force spectroscopy. It is quite oftenthat the solubility of the polyprotein is much less than the solubil-ity of the monomer and polyproteins often go into inclusion bodiesafter overexpression [13].
Fluorescence spectra
Fluorescence spectra of M-crystallin dimer in its native anddenatured 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 M-crystallin monomer insert (300 bp) and pQE80L vector (4800 bp). Lane 3, Doublerestriction digestion showing M-crystallin dimer insert (600 bp) and pQE80L vector(4800 bp).
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, purifiedM-crystallin dimer; lane 4, uninduced culture; lane 5, induced culture with 0.5 mMIPTG;
Fig. 4. Size exclusion chromatography results. M-crystallin dimer and monomer areeluted as single peaks (c) and (e), respectively. The molecular mass standards arealso 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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the protein at 295 nm. The fluorescence spectrum was collectedfrom 310 to 400 nm. The peak maximum was obtained at 332and 350 nm for the native (Fig. 5A) and the denatured states(Fig. 5B), respectively. The maximum arises from the fluorescenceof four Trp residues, two from each domain. Furthermore, the redshift in the fluorescence observed upon addition of 6 M guanidinehydrochloride as denaturant indicates that the Trp moiety gets sol-vent exposed, which was earlier in the core of protein. The fluores-cence properties of the dimer are identical to that of the monomer[6].
Circular dichroic spectra
M-crystallin dimer was characterized using far-UV and near-UVCD spectroscopy. Figs. 6A and 6B show far-and near-UV CD spectraof M-crystallin dimer, respectively. The far-UV CD spectrum showsminimum only at 214 nm without any minima at 208 or 222 nm,
indicating that the protein predominantly adopts b-sheet confor-mation, as in the case of monomer [6] and also as predicted byJPRED (Fig. 7A) [14]. Further, we have used a deconvolution meth-od (K2D2) proposed by Perez-Iratxeta et al. [15] to quantify thesecondary structural elements from the far-UV CD spectrum. Thismethod gave 5% of a-helix, 43% b-sheet and 52% random coil.These fractions are comparable with secondary structural elementscalculated from the NMR structure of M-crystallin monomer (36%b-sheet, and 64% of the random coil). Furthermore, the near-UV CDspectrum suggests that the aromatic amino acid residues are inasymmetric environments and the spectral features are very simi-lar to that of the monomer as reported by Barnwal et al. [16].
NMR characterization
The amino acid sequence of M-crystallin dimer is shown inFig. 7A. The 15N-labeled protein sample is prepared by growing
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 fluorescencespectrum 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.
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the transformed BLR(DE3) cells containing pQE80L-M-crystallin di-mer construct in a M9 minimal medium with 15NH4Cl as the solesource of nitrogen. The yield of protein is found to be 8 mg/L.The large spectral dispersion (3.5 ppm) of backbone amide 1Hchemical shifts, as seen in the sensitivity-enhanced 2D [15N–1H]-HSQC, indicates M-crystallin dimer is well folded as shown inFig. 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 inthe HSQC of M-crystallin monomer [16] in addition to correlationpeaks coming from the linker sequence and N-terminal (histidine)6
tag. For comparison, HSQC of monomer protein is also shown as anoverlay with that of the dimer. As is seen in the spectral overlay,the spectra of monomer and dimer look identical. This is anotherindication that the monomers in the dimeric protein have the same
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 2048data 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-squarebell 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.
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structure and that there is no interaction between the linker pep-tide and monomers. 1H, 13C and 15N resonance assignments of thedimer and the detailed structural characterization are underway.
Conclusion
In summary, we have synthesized a dimer of archaealM-crystallin linked in tandem with a decapeptide and character-ized its structure by optical and NMR spectroscopy. The structuralcharacteristics of the dimer shown by fluorescence, circular dichro-ism and the HSQC spectral analysis are similar to that of M-crystal-lin monomer. This indicates that the linker peptide does not affectthe structure of the protein. This methodology of linking proteinsin tandem could improve the current strategy used in makingpolyproteins that are regularly used in single molecule force spec-troscopy. As it would be impossible to do either NMR or crystallo-graphic structural studies on large polyproteins containing manyprotein units, our methodology of making dimer to aid its struc-tural characterization could bridge the structural characterizationof monomers in bulk to the mechanical stability studies onpolyproteins.
Acknowledgments
Authors would like to thank TIFR for financial assistance and thefacilities provided by the National Facility for High Field NMR,supported by DST, DBT, CSIR, New Delhi and TIFR, Mumbai.
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