Different expression levels of two KgmB-His fusion proteins

• Main Authors: Marković Sandra, Vojnović Sandra, Jovanović Milija Z., Vasiljević Branka Z.
• Format: Article
• Language: English
• Published: Serbian Chemical Society 2005-01-01
• Series: Journal of the Serbian Chemical Society
• Subjects: kgmb methylase; streptomyces tenebrarius; expression; purification
• Online Access: http://www.doiserbia.nb.rs/img/doi/0352-5139/2005/0352-51390512401M.pdf

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.