Cloning α and β chains of SLA-DR loci and reconstruction of their complex

Abstract

In order to investigate the prompt conformations of swine major histocompatibility complex (MHC), a swine MHC-II protein complex (SLA-II) was reconstructed in vitro. DRA and DRB were cloned from a crossbred commercial pig (Changbai-Dalan). Subcloned extracellular parts of DRA and DRB were linked together by a linker containing rich glycine/serine (G4S)3, and the whole length of two genes, named DRA-linker-DRB, was amplified by splicing overlap extension PCR (SOE PCR). DRA-linker-DRB was inserted into pMAL-p2X prokaryotic system and expressed. The expressed fusion protein MBP-DRA-(G4S)3-DRB was identified by Western-blot, purified and cleaved to obtain the protein of interest DRA-(G4S)3-DRB. The secondary structure of this protein was determined in circular dichroism (CD) apparatus. The results indicated that the subsequent protein MBP-DRA-(G4S)3-DRB was soluble and its molecule weight was 83.4 ku in consistent with the Western-blot. Cleaved by Factor Xa, the protein of interest was separated with a molecular weight of 40.9 ku. The CD spectrum demonstrated that the protein displayed a favorable ?-Helix structure, and the contents of ?-Helix, ?-sheet, turn, and random coil were 80 aa, 121 aa, 101 aa and 80 aa respectively. The identical ratios of ?-Helix, ?-sheet, turn, and random coil between MBP-DRA-(G4S)3-DRB and DRA-(G4S)3-DRB were 95%, 96.7%, 91.1% and 93.0%, respectively. The results also suggested that the reconstructed SLA-II complex presented an ideal conformation and can be used for studying its structure and function in vitro.

References

1. Bradford M N (1976). A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem, 72: 248–254. doi:10.1016/0003‐2697(76)90527‐3
2. Chen Y H, Yang J T, Martinez H M (1972). Determination of the secondary structuresof proteins by circular dichroism and optical rotary dispersion. Biochemistry, 11(22): 4120–4131. doi:10.1021/bi00772a015
3. Denkberg G, Cohen C J, Segal D, Kirkin A F, Reiter Y (2000). Recombinant humansingle-chain MHC-peptide complexes made from E. coli by in vitro refolding: functional single-chain MHC-peptide complexes and tetramerswith tumor associated antigens. Eur J Immunol, 30(12): 3522–3532. doi:10.1002/1521‐4141(2000012)30:12<3522::AID‐IMMU3522>3.0.CO;2‐D
4. Kapust R B, Waugh D S (1999). Escherichia coli maltose-binding proteinis uncommonly effective at promoting the solubility of polypeptidesto which it is fused. Protein Sci, 8(8): 1668–1674
5. Patrick M R, Buchbinder D, Jolley X, Dennett Buchanan R (1999). Incidence of inflammatory myopathies in Victoria,Australia, and evidence of spatial clustering. J Rheumatol, 26(5): 1094–1100
6. Srinivasan M, Domanico S Z, Kaumaya P T, Pierce S K (1993). Peptides of 23 residues or greater are required to stimulate a highaffinity class II-restricted T cell response. Eur J Immunol, 23(5): 1011–1016. doi:10.1002/eji.1830230504
7. Uger R A, Barber B H (1998). CreatingCTL targets with epitope-linked beta 2-microglobulin constructs. J Immunol, 160(4): 1598–1605
8. Wang J Y, Zhu S G, Xu C F (2002). Biochemistry. 3rd ed.Beijing: Higher Education Press, 197–252 (in Chinese)
9. Wang Y, Rubtsov A, Heiser R, White J, Crawford F, Marrack P, Kappler J W (2005). Usinga baculovirus display library to identify MHC class I mimotopes. Proc Natl Acad Sci USA, 102(7): 2476–2481. doi:10.1073/pnas.0409798102
10. White J, Crawford F, Fremont D, Marrack P, Kappler J (1999). Solubleclass I MHC with beta2-microglobulin covalently linked peptides: specificbinding to a T cell hybridoma. J Immunol, 162(5): 2671–2676
11. Xia C, Wu D, Wu W X, Wan J Q, Wang L, Yang T Y, Wang Q, Ning Y B (2005). Cloning and expression of interferon-alpha/gammafrom a domestic porcine breed and its effect on classical swine fevervirus. Vet Immunol Immunopathol, 104(1–2): 81–89. doi:10.1016/j.vetimm.2004.10.005
12. Yan R Q, Li X S, Yang T Y, Xia C (2005). Characterization of BF2 and beta(2)m in three Chinese chicken lines. Vet Immunol Immunopathol,108(3–4): 417–425. doi:10.1016/j.vetimm.2005.06.002
13. Yang D F, Zhu H F, Wang Z H, Shen G X, Tian D Y (2005). Construction of singlechain Fv antibody against transferrin receptor and its protein fusionwith alkaline phosphatase. World J Gastroenterol, 11(21): 3300–3303