title: Expression and purification of human MHC class I-related chain molecule B-α1 domain.
abstract: Major histocompatibility complex (MHC) class I-related chain A/B (MICA/B) is a type of stress-induced molecule that plays an important role in tumor surveillance. MICA/B shares a similar structure with MHC class I molecules, but MICA/B contains a closed cleft, not an open one, in its N-terminal alpha1 domain. The alpha 1 domain was believed to have no roles in antigen presentation, because the closed cleft provides limited space for binding with known molecules, and the cleft of MICA/B have been reported no known functions. To study the possible function of the cleft located in human MICA/B's alpha 1 domain, we attempted to express the human MICB-α1 (hMICB-α1) domain allele protein, which is approximately 20.5 kDa, by utilizing an Escherichia coli (E. coli) secretory pathway. Protein expression was accomplished through the phosphate-limited inducible promoter. After purification using ammonium sulfate precipitation, phenyl hydrophobic Sepharose, SP Sepharose and HisTrap affinity Sepharose, recombinant human MICB-α1 (rhMICB-α1) was obtained with 94.3% purity. The binding capacity of rhMICB-α1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-α1 can be prepared through the E. coli secretory pathway. Purified rhMICB-α1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-α1 protein, which can served as the basis for further studies of the possible function of the MICB cleft.
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Shufen
authors.initials: S
authors.lastname: Wang
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Zemin
authors.initials: Z
authors.lastname: Xiang
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Xu
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China. Electronic address: epazhang@outlook.com.
authors.forename: Dengyang
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China; College of Pu-er Tea, Yunnan Agricultural University, Kunming 650201, China. Electronic address: wangxuanjun@gmail.com.
authors.forename: Xuanjun
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Pu-er Tea Science, Ministry of Education, Yunnan Agricultural University, Kunming 650201, China; Agricultural Experiment Station for Tea and Tea Processing of Yunnan, Ministry of Agriculture, Kunming 650201, China; Tea Research Center of Yunnan, Kunming 650201, China. Electronic address: 623157535@qq.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Sheng
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015395:Histocompatibility Antigens Class I; C527085:KLRK1 protein, human; C098080:MICB antigen; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011994:Recombinant Proteins
keywords: MHC class I-related chain molecule B-α1 domain; Natural killer group 2- member D; Secretory expression