Biological characterisation and application of human MTH1 and monoclonal antibody preparation.

Biological characterisation and application of human MTH1 and monoclonal antibody preparation. Oncol Rep. 2018 Dec 19;: Authors: Chen C, Li X, Gu M, Di R, Kang N, Wang L, Lai Y, Liu Y, Ji X 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 (IC50) 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 (Km) and the catalytic constant (kcat) 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 immunofluores...
Source: Oncology Reports - Category: Cancer & Oncology Tags: Oncol Rep Source Type: research