Abstract:
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.
