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
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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.
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.
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.
KeywordsSwine leukocyte antigen 2 Crystal structure Foot-and-mouth disease virus Asia 1 serotype
Antigen presenting cells
Cytotoxic T lymphocyte antigen
Foot-and-mouth disease virus
Major histocompatibility complex
Swine leukocyte antigen
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  or autoantigen . 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 . Once CTLs are activated by the CTL epitopes, CTLs will recognize the epitopes and then kill the infected cells . 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 β2-microglobulin (β2m) 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 . 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) . There are three constitutively expressed classical and polymorphic SLA-I genes in the genome, namely SLA-1, SLA-2 and SLA-3 . Among them, SLA-2 is different from SLA-1 and SLA-3 in the N-terminal of their coding regions as previously described . The swine β2m (sβ2m) 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 . 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.
Expression and isolation of the proteins of SLA-2*HB01 and sβ2m
SLA-2*HB01-AS64-sβ2m complex information
E. coli (Rosetta)
Complete amino-acid sequence of the construct produced
Refolding and purification of the SLA-2*HB01-AS64-sβ2m complex
The refolded SLA-I (SLA-2*HB01-AS64-sβ2m) complex was carried out as described previously  with improvement recommended by Feng et al. . 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 sβ2m 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 24 h, the soluble SLA-2*HB01-AS64-sβ2m 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-sβ2m complex information is shown in Table 1.
Crystallizing the SLA-2*HB01-AS64-sβ2m complex
Sitting-drop vapor diffusion
Protein concentration (mg/mL)
Buffer composition of protein solution
50 mM NaCl, 20 mM Tris–HCl pH 8.0
Composition of reservoir solution
0.1 M BIS-TRIS pH 6.5, 0.2 M Ammonium acetate, 25% (w/v) PEG 3350
Volume and ratio of drop
1 μL protein solution mixed with 1 μL reservoir solution
Volume of reservoir (μL)
Data collection and processing
The SLA-2*HB01-AS64-sβ2m 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 . 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 .
X-ray diffraction data and processing statistics. Values in parentheses are for the highest resolution shell
ADSC Q315 CCD
Crystal-to-detector distance (mm)
Unit-cell parameters (Å)
a = 48.37, b = 97.75, c = 166.16
Resolution range (Å)
50.0–2.40 (2.40–2.49) a
Total No. of reflections
No. of unique reflections
Previously, we reported a multiple amino-acid sequence alignment between SLA-2*HB01 and other known MHC class I alleles in swine, human and mouse . Based on human HLA-A2 crystal data, the structure of SLA-2*HB01 was predicted by using homology modeling. We noticed SLA-2*HB01 preserved some key functional sites of HLA-A2 and H-2, which indicated that SLA-2*HB01 should be crucial in binding and presenting antigenic peptides [23, 24]. In addition, SLA-2*HB01 has 85.0–93.9, 86.2–97.0 and 83.3–88.6% sequence identity to other SLA-1, SLA-2 and SLA-3 alleles, respectively. Comprehensive analysis of SLA-2*HB01, it should be a novel allele of SLA-2 with specific genetic characteristics .
In this work, the crystal of the SLA-2*HB01 molecule complexed with swine β2m 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-sβ2m 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 . 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.
The research data will be used to further elucidate the 3-dimentional structure of the SLA-2 molecules and design more refined viral epitopes.
We thank Professor George F. Gao (Institute of Microbiology, Chinese Academy of Sciences) for providing experimental condition.
This work was supported in part by a grant from the National Natural Science Foundation of China (31672525), a grant from the National Natural Science Foundation of China (31172304), and a grant from the Liaoning Education Bureau Key Laboratory Project (LZ2015003).
Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
FSG designed, wrote, revised and submitted the manuscript. LF and PJ contributed to conduct the work, analysis and interpretation of results. ZBL, HG, XXZ and ZHZ contributed to the data collection and analysis. XH contributed to the manuscript revision. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
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