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Article

Effects of Lower Temperature on Expression and Biochemical Characteristics of HCV NS3 Antigen Recombinant Protein

1
Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan
2
Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30010, Taiwan
3
Department of Marine Environmental Engineering, National Kaohsiung University of Science and Technology, Kaohsiung City 81157, Taiwan
4
Department of Marine Biotechnology, National Kaohsiung Marine University, Kaohsiung 81143, Taiwan
*
Authors to whom correspondence should be addressed.
Academic Editors: Anwar Sunna and Jose M. Palomo
Catalysts 2021, 11(11), 1297; https://doi.org/10.3390/catal11111297
Received: 25 August 2021 / Revised: 18 October 2021 / Accepted: 26 October 2021 / Published: 27 October 2021
(This article belongs to the Special Issue Enzymes and Biocatalysis)
The nonstructural antigen protein 3 of the hepatitis C virus (HCV NS3), commonly-used for HCV ELISA diagnosis, possesses protease and helicase activities. To prevent auto-degradation, a truncated NS3 protein was designed by removing the protease domain. Firstly, it was overexpressed in E. coli by IPTG induction under two different temperatures (25 and 37 °C), and purified using affinity chromatography to attain homogeneity above 90%. The molecular mass of purified protein was determined to be approx. 55 kDa. While lowering the temperature from 37 to 25 °C, the yield of the soluble fraction of HCV NS3 was increased from 4.15 to 11.1 mgL−1 culture, which also improved the antigenic activity and specificity. The protein stability was investigated after long-term storage (for 6 months at −20 °C) revealed no loss of activity, specificity, or antigenic efficacy. A thermal stability study on both freshly produced and stored HCV NS3 fractions at both temperatures showed that the unfolding curve profile properly obey the three-state unfolding mechanism. In the first transition phase, the midpoints of the thermal denaturation of fresh NS3 produced at 37 °C and 25 °C, and that produced after long-term storage at 37 °C and 25 °C, were 59.7 °C, 59.1 °C, 55.5 °C, and 57.8 °C, respectively. Microplates coated with the fresh NS3 produced at 25 °C or at 37 °C that were used for the HCV ELISA test and the diagnosis outcome were compared with two commercial kits—Abbott HCV EIA 2.0 and Ortho HCV EIA 3.0. Results indicated that the specificity of the HCV NS3 produced fresh at 25 °C was higher than that of the fresh one at 37 °C, hence showing potential for application in HCV ELISA diagnosis. View Full-Text
Keywords: HCV; NS3; protein expression; diagnosis; helicase; protease HCV; NS3; protein expression; diagnosis; helicase; protease
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MDPI and ACS Style

Huang, C.-J.; Peng, H.-L.; Patel, A.K.; Singhania, R.R.; Dong, C.-D.; Cheng, C.-Y. Effects of Lower Temperature on Expression and Biochemical Characteristics of HCV NS3 Antigen Recombinant Protein. Catalysts 2021, 11, 1297. https://doi.org/10.3390/catal11111297

AMA Style

Huang C-J, Peng H-L, Patel AK, Singhania RR, Dong C-D, Cheng C-Y. Effects of Lower Temperature on Expression and Biochemical Characteristics of HCV NS3 Antigen Recombinant Protein. Catalysts. 2021; 11(11):1297. https://doi.org/10.3390/catal11111297

Chicago/Turabian Style

Huang, Chen-Ji, Hwei-Ling Peng, Anil K. Patel, Reeta R. Singhania, Cheng-Di Dong, and Chih-Yu Cheng. 2021. "Effects of Lower Temperature on Expression and Biochemical Characteristics of HCV NS3 Antigen Recombinant Protein" Catalysts 11, no. 11: 1297. https://doi.org/10.3390/catal11111297

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