TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21
| Main Authors: | Andi Yasmon; Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta 10320, Indonesia, Fera Ibrahim; Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta 10320, Indonesia, Budiman Bela; Department of Microbiology, Faculty of Medicine, University of Indonesia, Jakarta 10320, Indonesia |
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| Format: | Article application/pdf eJournal |
| Bahasa: | eng |
| Terbitan: |
Directorate of Research and Community Engagement, Universitas Indonesia
, 2011
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| Online Access: |
http://journal.ui.ac.id/index.php/science/article/view/741 |
Daftar Isi:
- Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, we reported effects of several conditions on purity and homogeneity of recombinant SARS-CoV N protein expressed in E. coli BL21. The SARS-CoV N gene was reverse transcribed and amplified by the reverse transcription-polymerase chain reaction (RT-PCR) technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the target gene into pQE-80L. After inserting the recombinant plasmid (pQE80-N) into E. coli, the recombinant protein (6 x His tag-N protein fusion) was expressed by inducing the bacterial cells with 0.1-0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) for 1-5 h. The protein recombinant were extracted from the bacterial cells by NTT buffer containing 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction of E. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mM imidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.