rRNA METHYLTRANSFERASES AND THEIR ROLE IN RESISTANCE TO ANTIBIOTICS rRNK METILTRANSFERAZE I NJIHOVA ULOGA U REZISTENCIJI NA ANTIBIOTIKE

MTaze metiluju nukleinske kiseline (DNK, RNK) i proteine, moduli{u}i tako njihovu aktivnost, funkciju i strukturnu organizaciju. Metilacija G1405 ili A1408 baza u 16S rRNK mikroorganizama koji proizvode aminoglikozide obezbe |u -je rezistenciju na sopstvene toksi~ne pro izvode. Ovaj mehanizam rezistencije je donedavno bio opi san samo kod proizvo|a~a antibiotika. Od 2003. godine i kod patogenih bakterija bele`i se neprestan porast rezi sten cije na aminoglikozide putem ovog mehanizma, {to predstavlja veliku pretnju efikasnoj upotrebi aminoglikozida u klini~koj praksi. Jedno od mogu}ih re{enja problema le`i u razvoju novih jedinjenja koja bi efikasno delovala na nova mesta u okviru ribozoma. Drugi pristup re{avanju ovog problema uklju~uje razvoj inhibitora MTaza odgovornih za rezisten ciju, sa idejom da se onemogu}i modifikacija bakte rijske rRNK i na taj na~in vrati terapeutska efikasnost postoje}im aminogliko zidima. Fundamentalna istra`ivanja vezana za proteinsku ekspresiju, potpuno razumevanje mehanizma rezistencije kao i razre{enje tercijarne strukture proteina su neophodan preduslov za primenu inhibitora 16S rRNK MTaza u medicini

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