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Troubleshooting in expression and purification of Recombinant Severe Acute Respiratory Syndrome-associated Coronavirus Nucleocapsid Protein in Escherichia coli BL21
Oleh:
Yasmon, Andi
;
Ibrahim, Fera
;
Bela, Budiman
Jenis:
Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi:
Makara Seri Sains vol. 14 no. 2 (Nov. 2010)
,
page 140-144.
Topik:
Imidazol
;
IPTG
;
N-lauroylsarcosine
;
Triton X
;
SARS-CoV N Protein
Ketersediaan
Perpustakaan Pusat (Semanggi)
Nomor Panggil:
MM65
Non-tandon:
1 (dapat dipinjam: 0)
Tandon:
tidak ada
Lihat Detail Induk
Isi artikel
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-Dgalactopyranoside (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.
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