Affiliations
- NMPA Key Laboratory for Quality Monitoring and Evaluation of Vaccines and Biological Products, 1 Sun Yat-Sen University, Guangzhou 510080, China; One Health Center of Excellence for Research and Training, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China; Key Laboratory of Tropical Diseases Control, Sun Yat-Sen University, Ministry of Education, Guangzhou 510080, China.
- NMPA Key Laboratory for Quality Monitoring and Evaluation of Vaccines and Biological Products, 1 Sun Yat-Sen University, Guangzhou 510080, China; One Health Center of Excellence for Research and Training, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, China; Key Laboratory of Tropical Diseases Control, Sun Yat-Sen University, Ministry of Education, Guangzhou 510080, China; Key Laboratory of Zoonose Prevention and Control at Universities of Inner Mongolia Autonomous Region, Medical College, Inner Mongolia Minzu University, Tongliao 028000, China; Key Laboratory of Livestock Infectious Diseases, Ministry of Education, Shenyang Agricultural University, Shenyang 110866, China. Electronic address: chenzliang5@mail.sysu.edu.cn.
PMID: 39304108 DOI: 10.1016/j.cca.2024.119967
Abstract
DNA synthesis is a critical process for cell growth and division. In cancer patients, an enzyme called thymidine kinase 1 (TK1) is often elevated in the blood, making it a valuable biomarker for cancer diagnosis and treatment. However, previous studies have shown that recombinant TK1 can exist in unstable mixtures of tetramers and dimers, leading to inconsistent results and potentially affecting accuracy. To address this issue, we hypothesized that incorporating tetrameric coiled-coil peptides could enhance TK1 self-assembly into stable tetramers without requiring additional adenosine triphosphate. In this study, we successfully expressed a recombinant TK1 tetramer protein in the Escherichia coli system. We optimized the induction conditions, significantly increasing protein expression levels, functionality, and solubility. Size exclusion chromatography confirmed the formation of a tetrameric structure in the expressed TK1 protein, with a molecular weight of 127.2 KDa, consistent with our expectations. We also found that the TK1 tetramer exhibited higher affinity with anti-TK1 IgY than wild-type TK1, as shown by enzyme-linked immunosorbent assay experiments. Moreover, the TK1 tetramer demonstrated good stability against heating, freeze-thawing and lyophilization with almost no immunoactivity lost. These findings suggest that recombinant TK1 tetramers have the potential to serve as calibrators in diagnostic assay kits, becoming promising candidates for quality control of clinical laboratory and in vitro diagnostic reagents.
Keywords: Biomarker; Self-assembly; Stability; Synthetic biology; Tetramer structure; Thymidine kinase 1.
