Construction of multiple recombinant SLA-I proteins by linking heavy chains and light chains in vitro and analyzing their secondary and 3-dimensional structures

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• 作者：Feng-shan Gao^a ; ¹ ; ^{gfsh0626@126.com} ; Jing Bai^a ; ¹ ; Qiang Zhang^b ; Chong-bo Xu^a ; Yanmin Li^c
• 关键词：3D ; 3-dimensional ; CD ; circular dichroism ; CTL ; cytotoxic lymphocyte ; MBP ; maltose binding protein ; MHC ; major histocompatibility complex ; PBS ; phosphate-buffered saline ; SOE-PCR ; splicing overlap extension PCR ; SLA ; swine leukocyte antigen
• 刊名：Gene
• 出版年：2012
• 期刊代码：73_03781119
• 类别：bio
• 出版时间：10 July, 2012
• 卷：502
• 期：2
• 页码：147-153
• 文件大小：1246 K

摘要

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 尾₂m 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-尾₂m 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-尾₂m 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 伪-helical structure, and the average 伪-helix, 尾-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.