Expression of the Non-structural Protein NSP10 and NSP16 of the SARS-CoV-2 Coronavirus in Eschericia coli Ita Saemena, Sari Dewi Kurniasih, Alfredo Kono, Yanti Rachmayanti, Zeily Nurachman*
Biochemistry Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung
Jl. Ganesha No. 10, Bandung - 40132, Indonesia
*Corresponding Author: zeily[at]itb.ac.id
Abstract
Coronavirus (SARS-CoV-2) is an RNA virus containing genes encoding 16 non-structural proteins (NSPs) that are very important for the virus life cycle. When this virus infects a host cell, the viral RNA must be protected from the host cell immune system. The NSP10 and NSP16 complexes act as methyltransferase enzymes in the coronavirus which are crucial for the virus replication process in the host cell. The potential of NSP10 and NSP16 to be used to protect RNA structures generally is not limited to the SARS-CoV-2 viral RNA. Therefore, this study aimed to clone and express NSP10 and NSP16 of SARS-CoV-2 coronavirus in Escherichia coli and to test their activity in increasing RNA stability. Research methodology includes assembly of synthetic NSP10 and NSP16 genes from sequences available in the NCBI database, constructing of recombinant NSP10 and NSP16 genes each with pET16b and pETDuet expression vectors, transfer of recombinant plasmids into E. coli BL21 and pLysS, production and purification of protein, and protein expression analysis by SDS-PAGE. The results showed that the recombinant NSP10-pET16b and NSP16-pET16b were overexpressed in E. coli BL21 host cells by induction of 1 mM IPTG. The two proteins have different expression levels at various temperatures, where NSP10 is better expressed at 18 C while NSP16 is at 37 C. Crude protein was successfully isolated from E. coli cells by a combination method of freeze-thaw lysis and sonication. Pure protein was obtained by purification using Ni-NTA matrix and elution using 450 mM imidazole, i.e. as much as 1.00 ug/uL for NSP10 and 2.01 ug/uL for NSP16. The study will continue on testing the activity of the NSP10-NSP16 protein complex as a methyltransferase for RNA.
