doiSerbia

Different expression levels of two KgmB-His fusion proteins

Marković Sandra (Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd)
Vojnović Sandra (Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd)
Jovanović Milija Z. (Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd)
Vasiljević Branka Z. (Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd)

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.

Keywords: KgmB methylase, Streptomyces tenebrarius, expression, purification