Original scientific paper Different expression levels of two KgmB-His fusion proteins

Abstract: The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His) 6 tag at the N-terminus, and pQEK-C, which places a (His) 6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His) 6 tag at the N-terminus showed a higher level of expression. Purification of the (His) 6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His) 6 tag at the N-terminus was purified to homogeneity>95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies

Original scientific paper Expression and purification of the Sgm protein from E. coli

Abstract: Thesgm gene from Micromonospora zionensis, the producer of the aminoglycoside antibiotic G-52, encodes for Sgm methylase which modifies the target site on 16S rRNAand thus protects the producer against its own toxic product. The sgm gene was modified 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 by Western blot analysis