Expression, purification, crystallization and preliminary X-ray diffraction analysis of swine leukocyte antigen 2 complexed with a CTL epitope AS64 derived from Asia1 serotype of foot-and-mouth disease virus

Background Currently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified. Results A complex of SLA-I constituted by an SLA-2*HB01 molecule with swine β2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64–72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å. Conclusion This research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.


Background
Class I of major histocompatibility complex (MHC) molecules are membrane-surface proteins, which are mainly responsible for binding and presenting endogenous antigenic peptides degraded from proteins coded by virus genome in target cells [1] or autoantigen [2]. These endogenous antigenic peptides usually constituted by 8-10 residues in length are derived from endogenous antigens degraded by cellular proteasome [3,4]. When a peptide is complexed and correctly refolded with MHC class I molecule in the antigen presenting cells (APCs), it will be presented to the membrane surface of the APCs to intrigue the cytotoxic T lymphocytes (CTLs). On this occasion, the peptide is defined as a CTL epitope [5]. Once CTLs are activated by the CTL epitopes, CTLs will recognize the epitopes and then kill the infected cells [6]. Therefore, the expressed MHC class I molecules on membrane of APCs should contain three components: a polymorphic heavy chain (α chain) of class I, a monomorphic light chain of β 2 -microglobulin (β 2 m) noncovalently linking the α3 domain of the heavy chain and an epitope bound in a groove formed by the α1 and α2 domains of the heavy chain [7]. Analysis of structural characteristics of MHC class I is necessary so that the mechanism of antigen presentation associated with MHC class I molecules will be explored.
MHC class I genes in pigs (Sus scrofa domestica) located in the 7p1.1 band of the short arm of chromosome 7 are also named as swine leukocyte antigen class I (SLA-I) [8]. There are three constitutively expressed classical and polymorphic SLA-I genes in the genome, namely SLA-1, SLA-2 and SLA-3 [9]. Among them, SLA-2 is different from SLA-1 and SLA-3 in the N-terminal of their coding regions as previously described [10]. The swine β 2 m (sβ 2 m) is monomorphic and noncovalently links with the heavy chain of the SLA-I molecules, which bind a viral or an auto CTL epitope. Foot-and-mouth disease virus (FMDV) is a great danger to cloven-hoofed animals including pigs, because it can cause animals to develop an acute, febrile, and highly contagious infectious disease [11]. In FMDV, there are seven serotypes named as A, O, C, Asia1, SAT1, SAT2, and SAT3. However, none of them have mutual cross-immunity [12,13]. Among them, the Asia1 serotype often occurs in Asian countries as previously reported [14,15]. Therefore, to further epitope vaccine development, more CTL epitopes and their interactions with SLA-I should be investigated. Recently, crystal data of the SLA-1, SLA-2 and SLA-3 had been announced, and a few CTL epitopes derived from swine-origin influenza virus, O serotype of FMDV, Ebola virus and respiratory syndrome virus (PRRSV) were also discovered [16][17][18][19]. However, crystal of SLA-2 associated with CTL epitope derived from Asia1 serotype of FMDV remains elusive.
In this article, we introduce the expression, refolding, purification, crystallization and preliminary X-ray diffraction analysis of SLA-2*HB01 with an AS64 CTL epitope derived from the Aisa1 serotype of FMDV.

Methods
Expression and isolation of the proteins of SLA-2*HB01 and sβ 2 m To construct the expression system of the SLA-2 haplotype HB01 allele (GenBank accession No. AB602431) coding for 275 amino acids in extracellular domain, a pair of primers was designed as shown in Table 1. The PCR product was recovered and cloned into pMD®19-T simple vector as previously described [10,19]. After identification by digestion with Nde I and Xho I and followed by sequencing, the interest of SLA-2*HB01 was further cloned into the pET21a (+) vector. The recombinant pET21a (+) containing the sβ 2 m had been done in our laboratory previously [19]. The two recombinant SLA-2*HB01 and sβ 2 m plasmids were induced to express in BL21 (Rosetta) Escherichia coli strain. The inclusion bodies were extracted as follows [17,19]: In 2 L Luria-Bertani medium (LB), the SLA-2*HB01 and sβ 2 m expression strains were inoculated and incubated at 180 rpm for 3-4 h in a shaking incubator at 37°C until the OD 600 value reached 0.5-0.6. Then, a final concentration of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was used in the medium to induce the interest of proteins to express in same cultivating environment as above. After 5 h, the bacteria were collected and cooled at 4°C for 30 min. Then the bacteria were centrifugated for 15 min at 6000 rpm at 4°C to collect pellets followed by washing them for three times with a solution buffer consisting of 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, 0.5% (v/v) Triton X-100, 1 mM DTT. In the end, a guanidinium chloride  Refolding and purification of the SLA-2*HB01-AS64-sβ 2 m complex The refolded SLA-I (SLA-2*HB01-AS64-sβ 2 m) complex was carried out as described previously [6] with improvement recommended by Feng et al. [19]. Firstly, the epitope AS64 (ALLRSATYY) derived from the VP1 protein residues 64-72 of Asia1 FMDV was firstly dissolved in dimethyl sulfoxide (DMSO) and then diluted in water. The inclusion body proteins of SLA-2*HB01 and sβ 2 m were renatured and refolded with the AS64 epitope according to a 1:1:3 molar ratio by using the gradual dilution method in a refolding buffer (100 mM Tris pH 8.0, 400 mM L-Arg HCl, 2 mM EDTA, 5 mM GSH, 0.5 mM GSSH, 0.5 mM PMSF) at 4°C. After refolding for 24h, the soluble SLA-2*HB01-AS64-sβ 2 m complex was further concentrated and purified by chromatography separation on a Superdex 200 16/60 HiLoad size-exclusion column (GE Healthcare) followed by an anion-exchange chromatography Resource Q (GE Healthcare). SLA-2*HB01-AS64-sβ 2 m complex information is shown in Table 1.
Crystallizing the SLA-2*HB01-AS64-sβ 2 m complex The final purified SLA-2*HB01-AS64-sβ 2 m complex proteins (~44 kDa) were further dialyzed against a crystallizing buffer (50 mM NaCl, 20 mM Tris-HCl pH 8.0) overnight at 4°C and then concentrated to 15 mg/mL. Crystallizing assays were tried with Index and Crystal Screen kit (Hampton Research) at 4°C and 18°C by using the sitting-drop vapor diffusion method as previously described [19,20]. Two protein solution drops each with 1 μl (at 7.5 and 15 mg/mL) and 1 μL of reservoir crystallizing buffer were placed and mixed together over a well containing 120 μL reservoir solution using an MRC Maxi plate (HR3-179, Hampton Research). Crystals of SLA-2*HB01-AS64-sβ 2 m were obtained at 14th day by using a protein concentration of 15 mg/mL in the Index solution No. 79 (0.2 M Ammonium acetate, 25% w/v PEG 3350, 0.1 M BIS-TRIS pH 6.5) at 4°C. Crystallizing condition is shown in Table 2.

Data collection and processing
The SLA-2*HB01-AS64-sβ 2 m crystal was firstly soaked in reservoir solution supplemented with 17% (v/v) glycerol as a cryoprotectant for several seconds and then flash-cooled in a nitrogen stream at − 173°C [21]. Data collection was carried out using an ADSC Q315 CCD detector at a wavelength of 1.00000 Å. By using beam line BL17U of the Shanghai Synchrotron Radiation Facility (Shanghai, China), the crystal was collected to 2.4 Å resolution. The raw data was indexed, integrated, corrected for absorption, scaled and merged using HKL-2000 [22].

Results
After purification to homogeneity by Superdex 200 16/60 HiLoad size-exclusion chromatography, it was shown that the heavy chain of SLA-2*HB01 was successfully refolded together with the light chain of sβ 2 m and the CTL epitope of AS64. The yield of the SLA-2*HB01-AS64-sβ 2 m complex is about 10%. It was shown four peaks in the chromatographic elution profile (Fig. 1a). The peak 1 should be aggregated heavy chains of SLA-2*HB01. The peaks 2-6 detected in lanes 2 to 6 with SDS-PAGE should be the refolded SLA-2*HB01-AS64-sβ 2 m complex (44 kDa). The peak 7 detected in lane 7 with SDS-PAGE should be surplus sβ 2 m (see inset in Fig. 1a). Another peak between peak 2-6 and peak 7, not shown in SDS-PAGE detection, should be some degraded proteins according to our previous practice. The refolded SLA-2*HB01-AS64-sβ 2 m complex was further purified by an anion-exchange chromatography Resource Q with a NaCl elution concentration of 21.5-26.5%. It was shown two peaks for the further purified complex (Fig. 1b). The peak 1 detected in lane 1 with SDS-PAGE contains free sβ 2 m. The peaks 2-4 detected in lanes 2 to 4 with SDS-PAGE contain two bands at the expected molecular weights of SLA-2*HB01 heavy chain (32 kDa) and sβ 2 m light chain (12 kDa) (see inset in Fig. 1b). The purified SLA-2*HB01-AS64-sβ 2 m was concentrated to 15 mg/mL for crystal screening. Crystals occurred after 7 days in the Index solution No. 79 at 4°C and then they were harvested at 14th d (Fig. 2). After preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the SLA-2*HB01-AS64-sβ 2 m crystal used for data collection was 2.4 Å. The space group of the crystal belongs to P2 1 2 1 2 1 . The unit-cell parameters of the crystal were shown as a = 48.37, b = 97.75, c = 166.163 Å (Fig. 3). The crystal has a solvent content of 61.9%. Data-collection and processing statistics are summarized in Table 3.
In this work, the crystal of the SLA-2*HB01 molecule complexed with swine β 2 m and a CTL epitope AS64 derived from the Asia1 serotype of FMDV was reported. It seems the space group type of SLA-2*HB01-AS64-sβ 2 m crystal is consistent with that of SLA-1 crystal but quite different from that of SLA-3 crystal, which indicates the structure of SLA-2 might be more similar to that of SLA-1 [17,18]. Recently, a crystal of SLA-2*HB01 complex associated with an Hu64 CTL epitope derived from O serotype of FMDV was reported. It was shown that the two crystals were similar in space group type, but different in unit-cell parameters [19]. To learn about the elaborate structural characteristics of SLA-2, especially the special characteristics that differ from SLA-1 and SLA-3, the 3-dimentional structure of the SLA-2 complex associated more CTL epitopes derived from swine-origin virus is required to be revealed as soon as possible.

Conclusions
The research data will be used to further elucidate the 3-dimentional structure of the SLA-2 molecules and design more refined viral epitopes.