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 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