title: Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures.
abstract: Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β(2)m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β(2)m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β(2)m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1 kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6 kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Life Science and Technology, Dalian University, Dalian, Liaoning 116622, PR China. gfsh0626@126.com
authors.forename: Feng-shan
authors.initials: FS
authors.lastname: Gao
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Chong-bo
authors.initials: CB
authors.lastname: Xu
authors.forename: Yanmin
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D002942:Circular Dichroism / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013552:Swine
chemical_list: D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; C438668:swine leukocyte antigen