Biological characterisation and application of human MTH1 and monoclonal antibody preparation

  • Authors:
    • Chuyuan Chen
    • Xiaoping Li
    • Mengyue Gu
    • Rongrong Di
    • Nannan Kang
    • Lei Wang
    • Yisheng Lai
    • Yu Liu
    • Xuemei Ji
  • View Affiliations

  • Published online on: December 19, 2018     https://doi.org/10.3892/or.2018.6942
  • Pages: 1851-1862
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


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 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.
View Figures
View References

Related Articles

Journal Cover

March-2019
Volume 41 Issue 3

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Chen C, Li X, Gu M, Di R, Kang N, Wang L, Lai Y, Liu Y and Ji X: Biological characterisation and application of human MTH1 and monoclonal antibody preparation. Oncol Rep 41: 1851-1862, 2019
APA
Chen, C., Li, X., Gu, M., Di, R., Kang, N., Wang, L. ... Ji, X. (2019). Biological characterisation and application of human MTH1 and monoclonal antibody preparation. Oncology Reports, 41, 1851-1862. https://doi.org/10.3892/or.2018.6942
MLA
Chen, C., Li, X., Gu, M., Di, R., Kang, N., Wang, L., Lai, Y., Liu, Y., Ji, X."Biological characterisation and application of human MTH1 and monoclonal antibody preparation". Oncology Reports 41.3 (2019): 1851-1862.
Chicago
Chen, C., Li, X., Gu, M., Di, R., Kang, N., Wang, L., Lai, Y., Liu, Y., Ji, X."Biological characterisation and application of human MTH1 and monoclonal antibody preparation". Oncology Reports 41, no. 3 (2019): 1851-1862. https://doi.org/10.3892/or.2018.6942