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[Cloning, expression and purification of silent information regulator 2 from Giardia lamblia]
Xia Zhang1, Hong-Mei Ju, Yun-Hua Wang
1Medical School, Dalian Uninversity, Dalian 116622, China.
Objective:
To clone and express silent information regulator 2 (Sir2) gene from Giardia lamblia.
Methods:
The GlSir2 gene was amplified by PCR from genomic DNA of Giardia lamblia (Chinese strain C2 clone). PCR product was cloned into pMD-19T vector and transformed into E coli JM109. The recombinant plasmid was sequenced and then cloned into the pET28b vector. The pET28b-Gl1Sir2 recombinant plasmid was transformed into E. coli BL21(DE3), followed by expression of the protein induced by IPTG. The recombinant protein was analyzed by SDS-PAGE. Inclusion bodies were dissolved with 8 mol/L urea, and the supernatant was collected and applied to Ni2+ affinity chromatography. The purified recombinant protein was renatured by dialysis and verified by Western blotting using anti-His tag antibody.
Results:
GlSir2 gene sequence was cloned. The GISir2 open reading frame (1 680 bp) encoded a 559-amino acid protein with Mr 62 800. The recombinant plasmid pET28b-GlSir2 expressed an inclusion body protein of GISir2 after being induced with IPTG. The protein purity reached above 80% after purification. The purified protein was renatured by dialysis. The recombinant GISir2 was recognized by anti-His tag antibody.
Conclusion:
The coding sequence of GLSir2 gene was cloned and expressed in vitro. The recombinant protein was identified by anti-His tag antibody.
