Sains Malaysiana 52(12)(2023): 3395-3405

http://doi.org/10.17576/jsm-2023-5212-04

Characterisation of Recombinant 3CL Protease from SARS-CoV-2 Produced in E. coli BL21 (DE3) for Screening Anti-Covid Drug Candidates using Rhodamine 110-Synthetic Peptide Conjugate as a Substrate

 (Pencirian Protease 3CL Rekombinan daripada SARS-CoV-2 Dihasilkan dalam E. coli BL21 (DE3) untuk Menyaring Calon Dadah Anti-Covid menggunakan Rhodamine 110-Sintetik Peptida Konjugat sebagai Substrat)

I GEDE EKA PERDANA PUTRA¹, MARIA ULFAH¹, NAUFAL HAFIZH², ERWAHYUNI ENDANG PRABANDARI³, FIRDAYANI³ & IS HELIANTI^1,*

  ¹Research Center for Applied Microbiology, National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

²Research Center for Agroindustry, National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

³Research Center for Vaccine and Drug,National Research and Innovation Agency, Jalan Raya Bogor Km 46, Cibinong, Bogor, Indonesia

Received: 5 April 2023/Accepted: 7 November 2023

Abstract

The prediction that the pandemic is progressing towards becoming endemic does not change the fact that COVID-19 can still be fatal for individuals with weak immune systems. Therefore, anti-COVID drugs are still needed, even when the disease becomes endemic. With regards to SARS-CoV-2, the roles of 3CL protease are crucial in the formation of new virus particles. Therefore, inhibiting the function of these viral proteases will directly prevent viral replication in the human body. In this study, we report the production of a recombinant 3CL protease from SARS-CoV-2 in E. coli BL21 (DE3), which has not been extensively studied in Indonesia. The purified 3CL protease exhibited high solubility and functional activity. Additionally, the recombinant enzyme was characterised using the Rhodamine 110 fluorogenic peptide substrate. We showed that the recombinant 3CL protease was unstable in the presence of a DMSO concentration above 10%. Using the Rhodamine 110 fluorogenic peptide substrate, we found that the enzyme had a K_M of 47.0 µM, V_max of 0.41 RFU/s, and k_cat/K_M of 0.0088 RFU/μM²_.s while the IC₅₀ of the GC376 was 13.35 nM. We also tested three bioactive compounds (catechin, emodin, and 1,4-naphthoquinone) using this recombinant protease as a protein target, and 1,4-naphthoquinone was the most promising bioactive compound in inhibiting the SARS-CoV-2 virus.

Keywords: Characterisation; peptide substrate LGSAVLQ-Rh110; recombinant 3CL protease

Abstrak

Ramalan bahawa wabak itu sedang berkembang menjadi endemik tidak mengubah fakta bahawa COVID-19 boleh membawa maut bagi individu yang mempunyai sistem imun yang lemah. Oleh itu, dadah anti-COVID masih diperlukan, walaupun penyakit itu menjadi endemik. Berkenaan dengan SARS-CoV-2, peranan 3CL protease adalah penting dalam pembentukan zarah virus baharu. Oleh itu, merencat fungsi protease virus ini secara langsung akan menghalang replikasi virus dalam tubuh manusia. Dalam kajian ini, kami melaporkan penghasilan 3CL protease rekombinan daripada SARS-CoV-2 dalam E. coli BL21 (DE3), yang belum dikaji secara meluas di Indonesia. 3CL protease yang telah ditulenkan menunjukkan keterlarutan yang tinggi dan aktiviti berfungsi. Tambahan lagi, enzim rekombinan telah dicirikan menggunakan substrat peptida fluorogenik Rhodamine 110. Kami mendapati bahawa 3CL protease rekombinan ini tidak stabil dalam kepekatan DMSO melebihi 10%. Dengan menggunakan substrat peptida fluorogenik Rhodamine 110, kami mendapati bahawa enzim ini mempunyai nilaiK_M 47.0 µM, V_max 0.41 RFU/s dan k_cat/K_M 0.0088 RFU/μM²_.s manakala nilai IC₅₀ GC376 ialah 13.35 nM. Kami juga menguji tiga sebatian bioaktif (katechin, emodin dan 1,4-naftoquinon) menggunakan protease ini sebagai sasaran protein dan 1,4-naftoquinon didapati adalah sebatian bioaktif yang paling berpotensi dalam menghalang virus SARS-CoV-2.

Kata kunci: Pencirian; rekombinan 3CL protease; substrat peptida LGSAVLQ-Rh110

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*Corresponding author; email: is.helianti@brin.go.id