Cloning and Expression of SARS-CoV-2 Membrane Recombinant Protein in Prokaryotic Expression System

Abstract

Background: The M protein is one of the structural protein of SARS CoV-2. The M protein is relatively conserved and stable than other structural protein. This made immunodominant epitopes of M protein has the advantage to be learned with the aim to understanding its immunogenicity and its antigenicity. Several studies have shown that M protein successfully expressed in several expression system, E. coli was one of them. In this study, M gene of SARS CoV-2 was cloned, sequenced, expressed in E. coli BL21 (DE3) system, and purified with denature condition. The membrane recombinant protein can be used for development of a SARS CoV-2 antibody diagnostic system.Methods: the gene encoding the SARS-CoV-2 M protein in the form of gBlocks was cloned into the cloning vector and then subcloned into the pQE80L prokaryotic expression vector. There were three stages of recombinant plasmids verification which were the colony PCR, restriction, and sequencing. The M gene cloned in pQE80L was expressed by using BL21 and purified under denature condition.Results: The recombinant plasmid pQE80L was confirmed containing M protein using primer that specifically amplify the multiple cloning sites (MCS) of pQE80L and produce 595 bp amplicon that indicating the presence of recombinant gene. Restriction of recombinant plasmid using BamHI and HindIII produced 306 bp and 4709 bp DNA bands. The sequence of M gene in pEQ80L has been confirmed by sequencing. Further to ensure the M gene could be expressed in prokaryotic system, the recombinant plasmid was transformed into BL21 bacteria. The SARS-CoV-2 membrane protein with a size of 11,83 kDa has been successfully expressed and purified using the Ni-NTA agarose purification technique under denature conditions.Conclusion: the gene encoding the membrane protein of SARS-CoV-2 has been successfully cloned and expressed in the prokaryotic expression system. AbstrakLatar belakang: Protein M merupakan salah satu protein struktural SARS-CoV-2. Protein M merupakan protein yang relatif lestari dan tidak mudah untuk bermutasi dibandingkan protein struktural lainnya. Hal ini menjadikan protein M memiliki kelebihan untuk dipelajari epitop imunodominannya, dengan tujuan untuk memfasilitasi pemahaman terkait imunogenisitas dan antigenisitasnya. Beberapa penelitian menunjukkan ekspresi protein M telah dilakukan pada beberapa sistem ekspresi, salah satunya pada sistem ekspresi E. coli. Pada penelitian ini dilakukan pengklonaan gen M SARS CoV-2, sekuensing, ekspresi pada E. coli BL21(DE3), dan purifikasi dengan kondisi denatur. Protein rekombinan membran ini kemudian dapat digunakan dalam pengembangan sistem diagnostik antibodi SARS CoV-2.Metode: gen penyandi protein M SARS-CoV-2 dalam bentuk gBlocks diklona ke dalam vector pengklonaan dan kemudian disubklona ke dalam vector ekspresi prokariot pQE80L. Tiga tahapan verifikasi terhadap plasmid rekombinan, yaitu PCR koloni, restriksi, dan sekuensing dilakukan untuk memastikan bahwa gen target telah berhasil diklona. Plasmid ekspresi pQE80L yang membawa gen M kemudian diekspresikan dan dipurifikasi pada kondisi denatur. Hasil: Plasmid rekombinan pQE80L dikonfirmasi mengandung protein M menggunakan primer yang secara spesifik mengamplifikasi multiple cloning site (MCS) pQE80L dan menghasilkan amplicon berukuran 595 bp yang menunjukkan keberadaan gen rekombinan. Restriksi plasmid rekombinan menggunakan BamHI dan HindIII menghasilkan pita DNA berukuran 306 bp dan 4709 bp. Sekuen genM di pQE80L dikonfirmasi dengan sekuensing. Untuk memastikan bahwa gen M dapat diekspresikan di sistem prokariot, plasmid rekombinan ditransformasi ke dalam bakteri BL21. Protein membran SARSCoV-2 dengan ukuran 11,83 kDa telah berhasil diekspresikan dan dipurifikasi menggunakan teknik purifikasi Ni-NTA agarose pada kondisi denature.Kesimpulan: gen penyandi protein membrane SARS-CoV-2 telah berhasil diklona dan diekspresikan pada sistem ekspresi prokariot