Expression and purification of the Sgm protein from E. coli

Abstract

The sgm gene from Micromonospora zionensis, the producer of the aminoglycoside antibiotic G-52, encodes for Sgm methylasewhich modifies the target site on 16S rRNA and thus protects the producer against its own toxic product. The sgm gene wasmodified by polymerase chain reaction (PCR) and cloned in the QIAexpress pQE-30 vector in order to make a construct that places the (His)6 tag at the N-terminus of the protein. The resulting expression construct was transformed in the E. coli strain NM522 and the functional activity of the Sgm-His fusion protein was confirmed in vivo. Purification of the (His)6-tagged Sgm protein by Ni-NTA affinity chromatography was performed under native conditions and the protein was detected on a sodium dodecyl sulfate polyacrylamide gel. Sgm methylase was purified to homogeneity > 95 %. Polyclonal antibodies raised to purified (His)6-tagged Sgm protein were used to identify this protein byWestern blot analysis.

Keywords

• Sgm methylase
• Micromonospora zionensis
• expression
• E. coli
• purification