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ArtikelTroubleshooting 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 artikelConsidering 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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