Expression, crystallization and preliminary crystallographic analysis of C-reactive protein from zebrafish

Crystals of native and selenomethionine-substituted C-reactive protein from zebrafish diffracted to 2.3 and 1.7 Å resolution, respectively, and belonged to space group R3 with one molecule per asymmetric unit. The Matthews coefficient was calculated to be 3.28 ų Da⁻¹.

Abstract

C-reactive protein (CRP) is an acute phase protein that is found in blood, the concentration of which in plasma rises rapidly in response to inflammation. It functions as a pattern-recognition molecule, recognizing dead cells and various pathogenic agents and eliminating them by utilizing the classical complement pathway and activating macrophages. CRP is phylogenetically highly conserved in invertebrates and mammals. To date, information on the CRP gene has been reported from numerous species of animals, but little is known about the structure of CRP from species other than humans. In order to solve the structure of CRP from bony fish, the CRP gene from zebrafiah (Danio rerio) was cloned and expressed in Escherichia coli. The zebrafish CRP (Dare-CRP) was then purified and crystallized. The crystal diffracted to 2.3 Å resolution and belonged to space group R3, with unit-cell parameters a = b = 114.7, c = 61.0 Å. The Matthews coefficient and solvent content were calculated to be 3.28 ų Da⁻¹ and 62.55%, respectively. Determination of the zebrafish CRP structure should be helpful in investigating the evolution of CRPs in the innate immune system.

1. introduction

C-reactive protein (CRP) is an ancient protein that has been studied for about 80 years since its discovery in 1930 in Oswald Avery’s laboratory (Tillett & Francis, 1930 ▶). CRP is typically comprised of five identical subunits that are held together by noncovalent forces (Shrive et al., 1996 ▶). It was originally defined as an acute phase protein as its concentration increases immediately and dramatically in the blood during the early stages of inflammation. Presently, as a member of the classical pentraxins, accumulated studies on CRP have shed light on the role of this molecule in various important disease processes such as cardiovascular, infectious and autoimmune diseases (Agrawal et al., 2009 ▶).

CRP genes have been found in almost all animals from the arthropod Limulus to humans. It is interesting to note that the primary sequences of different animal CRPs share low identity. In Limulus CRP displays obvious polymorphism (Iwaki et al., 1999 ▶), but this phenomenon is not found in CRPs from humans or other mammals. In addition to the classical pentamer, the oligomeric form of different CRPs can also vary (Eisenhardt et al., 2009 ▶; Shrive et al., 2009 ▶). Furthermore, mammalian CRPs possess a number of other important properties. For example, CRP serves as a pattern-recognition molecule that recognizes pathogenic agents and binds to C1q and Fcγ receptors (FcγRs) to eliminate these pathogens (Lu et al., 2008 ▶). Although C1q and FcγRs genes have been identified from primal fish to humans, it is still unknown whether fish CRP can bind these two molecules. Here, we report the cloning, expression, purification and crystallization of recombinant CRP (Dare-CRP) from zebrafish (Danio rerio).

2. Materials and methods

2.1. Cloning and expression

The CRP gene was amplified from a D. rerio cDNA library using Ex Taq DNA polymerase [Takara Biotechnology (Dalian) Co. Ltd]. After the amplification of the target gene with the sense primer 5′-­CGGAATTCTTTAAAAATCTGAGCGGTAAAGTG-3′ and the antisense primer 5′-CCGCTCGAGTTATCAGTTATCTGGAACC­ACAAGCA-3′, the PCR product was digested with the restriction enzymes NdeI and XhoI and then inserted into the pET21a vector (Novagen, Merck KGaA, Darmstadt, Germany). Recombinant Dare-CRP protein and its selenomethionine-substituted form were expressed in the form of inclusion bodies in Escherichia coli strain BL21 (DE3) and the methionine-auxotrophic E. coli strain B834 (DE3) (Blackburn et al., 1999 ▶), respectively (Novagen, Merck KGaA, Darmstadt, Germany). When the OD₆₀₀ reached about 0.6, 2 mM isopropyl β-d-1-thio­galactopyranoside (IPTG) was added to both media. After 8 h, the cells were harvested by centrifugation and lysed by sonication. Pure inclusion bodies were obtained by washing the pellet three times with a solution consisting of 20 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.5% Triton X-100 (Garboczi et al., 1996 ▶; Zhang et al., 2010 ▶). The inclusion bodies were dissolved to a con­centration of 30 mg ml⁻¹ in a buffer consisting of 6 M guanidine–HCl (Gua–HCl), 50 mM Tris–HCl pH 8.0, 10 mM EDTA, 100 mM NaCl, 10%(v/v) glycerine and 10 mM DTT (Zhang et al., 2010 ▶).

2.2. Comparison of the Dare-CRP sequence with those of CRPs from other species

Multiple sequence alignment of CRPs from different species was performed using the ClustalW2 program (Fig. 1 ▶). Identical amino acids are shown as white text on a red background, while similar amino acids are shown as red text on a white background. Alignment scores between the Dare-CRP sequence and the other sequences are shown at the ends of the sequences. The sequences aligned are as follows: zebrafish (GenBank accession No. JF772178), Limulus (GenBank accession No. AAA28270), chicken (GenBank accession No. ABD16281), mouse (GenBank accession No. CAA31928) and human (GenBank accession No. AAA52075).

Figure 1.

Multiple sequence alignment of CRPs from different species. The sequence alignment was performed using ClustalW2. The sequences aligned are as follows: zebrafish (GenBank accession No. JF772178), Limulus (GenBank accession No. AAA28270), chicken (GenBank accession No. ABD16281), mouse (GenBank accession No. CAA31928) and human (GenBank accession No. AAA52075). Identical amino acids are shown in white text on a red background, while similar amino acids are shown in red text on a white background. The alignment scores between the Dare-CRP sequence and the other sequences are shown at the end of the sequences.

2.3. Refolding and purification of the Dare-CRP protein

The preparation of native and selenomethionine-substituted Dare-CRP protein was essentially carried out as described previously (Garboczi et al., 1996 ▶) with modifications in our laboratory (Zhang et al., 2010 ▶). Briefly, the Dare-CRP inclusion bodies were dissolved in a solution consisting of 6 M Gua–HCl, 50 mM Tris–HCl pH 8.0. Dare-CRP was refolded by the gradual dilution method. After 12 h incubation at 277 K, the soluble Dare-CRP protein was concentrated and purified using a Superdex 200 size-exclusion column (GE Healthcare; Zhang et al., 2010 ▶).

2.4. Crystallization

For crystallization, purified Dare-CRP protein was dissolved at concentrations of 2.5 and 6 mg ml⁻¹ in 50 mM NaCl, 20 mM Tris–HCl pH 8.0. Screening was carried out with the Index, Crystal Screen, Crystal Screen 2, Crystal Screen Cryo and Crystal Screen 2 Cryo kits (Hampton Research, California, USA; McFerrin & Snell, 2002 ▶). The sitting-drop vapour-diffusion method was used for crystal growth at 296 K. Drops were prepared by mixing 1 µl protein solution with 1 µl reservoir solution and were equilibrated against 150 µl of the same reservoir solution. Native and selenomethionine-substituted Dare-CRP crystals appeared after 7 d in condition No. 39 [0.085 M Na HEPES pH 7.5, 1.7%(v/v) polyethylene glycol 400, 1.7 M ammonium sulfate, 15%(v/v) glycerol] of Crystal Screen Cryo without any optimization.

2.5. Data collection and processing

Diffraction data for native Dare-CRP were collected on an R-­AXIS IV⁺⁺ image-plate detector using a Rigaku rotating-anode X-­ray generator with a radiation wavelength of 1.5418 Å. Data for selenomethionine-substituted Dare-CRP were collected on beamline NE3A at the KEK synchrotron facility (Tsukuba, Japan) at a wavelength of 1.0 Å using an ADSC Q270 imaging-plate detector. Both native and selenomethionine-substituted Dare-CRP crystals were cryoprotected by adding 33%(v/v) glycerol. The data were processed and scaled with HKL-2000 (Otwinowski & Minor, 1997 ▶). Selenomethionine-substituted Dare-CRP crystals diffracted to 1.7 Å resolution, while the native crystals diffracted to 2.3 Å resolution. Structure determination will be carried out using the multiple-wavelength anomalous diffraction (MAD) method.

3. Results and discussion

A multiple sequence alignment of CRP amino-acid sequences from GenBank was carried out with ClustalW2 (Fig. 1 ▶). According to the results of multiple sequence alignment, Dare-CRP shares the lowest identity with Limulus CRP and resembles mammalian CRPs more closely than avian CRP. Although CRPs exist in invertebrates and vertebrates, they share relatively low similarity (19–33%).

It is often the case that proteins that are not correctly folded will appear in the form of precipitation, aggregation etc. Incorrectly folded proteins are unstable and always remain insoluble during the course of refolding. In contrast, correctly refolded protein exists as a monomer, dimer or higher order oligomer, has good solubility and often maintains good stability. The refolding efficiency of Dare-CRP inclusion bodies was about 5%. According to the size-exclusion chromatography profile (Fig. 2 ▶) and the molecular weight of the Dare-CRP monomer (23.5 kDa), soluble Dare-CRP exists mainly as a dimer, with a small proportion existing as a higher order oligomer (corresponding to the main peak and the small peak, respectively, in Fig. 2 ▶). One possible reason for this might be that our Dare-CRP was produced in E. coli, whereas classical human CRP, which was directly obtained from serum, exists in its native conformation as a pentamer (Shrive et al., 1996 ▶). Another possibility is that the dimeric form of Dare-CRP is one form of native CRP in zebrafish, as monomeric, hexameric, heptameric and octameric pentraxins have been reported for different species (Eisenhardt et al., 2009 ▶; Shrive et al., 2009 ▶). The successful production of soluble CRP using a bacterial expression system was first reported by Dortay et al. (2011 ▶), thus providing a new way of obtaining CRP. The function of this dimeric Dare-CRP will be reported in due course.

Figure 2.

Purification of Dare-CRP. The gel-filtration profile of refolded Dare-CRP on Superdex G-200 FPLC. The insert shows the reduced SDS–PAGE gel (15%) of the corresponding purified protein; lane M contains molecular-weight markers (labelled in kDa) and lane S contains the purified protein sample.

Optimal Dare-CRP crystals appeared after 7 d (Fig. 3 ▶). The selenomethionine-subsitituted crystals diffracted to a maximum resolution of 1.7 Å, whereas the native crystals diffracted to 2.3 Å resolution (Fig. 4 ▶). Both crystals belonged to space group R3. The Matthews coefficient was calculated to be 3.28 ų Da⁻¹ (Table 1 ▶). There are three methionine residues in the Dare-CRP sequence and we intend to determine the structure of Dare-CRP using the multiple-wavelength anomalous diffraction (MAD) method. To date, only structures of human CRP (PDB entry 1gnh; Shrive et al., 1996 ▶) and of Limulus serum amyloid P component (SAP; a highly identical classical pentraxin; PDB entry 3flt; Shrive et al., 2009 ▶) have been reported. The monomer structures of human CPR and Limulus SAP are both comprised of two main β-sheets, one α-helix and several coils. Human CRP exists in both pentameric and monomeric forms, whereas Limulus SAP is reported to form a wider range of oligomers including hexamers, heptamers and octamers. In the absence of any classical pentraxin structures from intermediate species such as bony fish, our results should prove helpful in order to investigate the evolution of pentraxins in the animal kingdom.

Figure 3.

Typical crystals of native Dare-CRP.

Figure 4.

A typical diffraction pattern from a native Dare-CRP crystal.

Table 1. X-ray diffraction data and processing statistics of the refined structure.

Values in parentheses are for the highest resolution shell.

	Native	Selenomethionine
Space group	R3	R3
Unit-cell parameters (Å)	a = 114.7, b = 114.7, c = 61.0	a = 114.7, b = 114.7, c = 60.9
Resolution range (Å)	50–2.3 (2.38–2.30)	50–1.7 (1.76–1.70)
Total No. of reflections	76884	342067
No. of unique reflections	12957	32376
Completeness (%)	99.2 (93.1)	98.1 (93.5)
Average I/σ(I)	25.4 (5.3)	30.6 (5.2)
Rmerge† (%)	6.9 (25.8)	6.8 (28.7)
Average multiplicity	5.9 (5.3)	10.6 (9.0)

^† R _merge = , where I_i(hkl) is the observed intensity and 〈I(hkl)〉 is the average intensity from multiple measurements.