TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21

TROUBLESHOOTING IN EXPRESSION AND PURIFICATION OF RECOMBINANT SEVERE ACUTE RESPIRATORY SYNDROME-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN IN Escherichia coli BL21

Budiman Bela, Fera Ibrahim, Andi Yasmon

Considering importance of N protein for study of viral pathogenesis or development of immunodiagnostic assay, wereported 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 chainreaction (RT-PCR) technique. The amplicons were cloned into pGEX-6P1 and followed by subcloning of the targetgene into pQE-80L. After inserting the recombinant plasmid (pQE80-N) into E. coli, the recombinant protein (6 x Histag-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 buffercontaining 0-20 mM imidazol, and followed by Ni-NTA affinity resin purification. The results showed that induction ofE. coli BL21 with 0.2 mM IPTG for 4 h and followed with lysis of bacterial cells in NTT buffer containing 10 mMimidazol were optimal conditions to obtain the pure recombinant SARS-CoV N protein.

2010

İmidazol, IPTG, N-lauroylsarcosine, Triton X, SARS-CoV N protein

DOI:

Makara Seri Sains

Universitas Indonesia

Science (General)

EN