Efficient In Vitro Refolding and Characterization of Major Histocompatibility Complex Class I-Related Chain Molecules A (MICA) and Natural Killer Group 2 Member D (NKG2D) Expressed in E. coli.
Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.
Xin
X
Zhao
Desmond Omane
DO
Acheampong
Youfu
Y
Wang
Mingying
M
Tang
Wei
W
Xie
Zhiguo
Z
Chen
Min
M
Wang
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 154#, Tong Jia Xiang 24, Nanjing 210009, P.R. China. juancpu@126.com.
Juan
J
Zhang
D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D058849:Protein Refolding; D012995:Solubility
D015395:Histocompatibility Antigens Class I; C527085:KLRK1 protein, human; C099187:MHC class I-related chain A; D055655:NK Cell Lectin-Like Receptor Subfamily K