Biological characterisation and application of human MTH1 and monoclonal antibody preparation

Abstract

Human MutT homolog 1 (MTH1) hydrolyses oxidised nucleotide triphosphates, thereby preventing them from being incorporated into DNA; MTH1 has been found to be elevated in many types of cancers, including lung, stomach cancer, melanoma and breast cancer. Thus, tumour-targeted hMTH1 may be valuable for developing novel anticancer therapies. In the present study, we prepared human MTH1 protein and its monoclonal antibody (mAb). The hMTH1 gene was cloned into the prokaryotic expression vector pET28a and optimally expressed in the E. coli Transetta (DE3) strain. Using an Ni-NTA column and a G-50 gel filtration column, 20.1 mg of active hMTH1 was obtained from 1,000 ml of bacterial culture, and the purity was over 98%, as detected by high-performance liquid chromatography (HPLC). The half maximal inhibitory concentration (IC 50 ) of TH287 (hMTH1 inhibitor) was determined to be 3.53±0.47 nM using the recombinant hMTH1 protein (rhMTH1). The enzyme activity assay showed the Michaelis constant (K m ) and the catalytic constant (k cat ) of the protein were 106.13±48.83 µM and 3.64±0.58 sec -1 , respectively. The anti-hMTH1 mAb was obtained via the hybridoma technique and validated by western blot analysis. In addition, an immunofluorescence assay (IFA) and ELISA determined that the mAb could efficiently bind to natural hMTH1 expressed on the human breast cancer cell line MCF-7. Taken together, the results showed the rhMTH1 is an active protein and has practical applications for inhibitor selection, and our prepared hMTH1 mAb will provide a valuable tool for the further characterisation of hMTH1 and antitumour medicinal development in future.

Preparation of an anti-NEK2 monoclonal antibody and its application in liver cancer

“…The subsequent procedures were consistent with the protocol of the iELISA above. The calculation method of K aff was the same as that reported in a previous study [ 15 ].…”

Optimised expression and purification of RBP4 and preparation of anti‐RBP4 monoclonal antibody

“…According to the checkerboard square method, the RBP4 recombinant protein was coated on the 96-well microplate at the concentration of 4, 2, 1 and 0.5 lgÁmL À1 , and the selected mAb was serially diluted with a double dilution ratio (1 : 1000-1 : 1,024,000). The calculation method of affinity constant Ka was the same as that reported in the previous study [15]. Recombinant RBP4-His protein, recombinant sex hormone-binding globulin-His (SHBG-His) protein, recombinant serum amyloid A-4-His (SAA4-His) protein, recombinant never in mitosis gene-A related expressed kinase 2-His (NEK2-His) protein and BSA albumin were coated on the 96-well microplate at a concentration of 2 lgÁmL À1 , and the sample diluent was used as a negative control.…”