Zbornik - Društvo genetičara Srbije
Zbornik - Društvo genetičara Srbije
Zbornik - Društvo genetičara Srbije
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III-Usm-2 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE 69<br />
Subotica, 30. novembar - 4. decembar 2004.<br />
ANALIZA SKRAÆENOG KgmB PROTEINA IZ S. tenebrarius<br />
Sandra Markoviæ, Sandra Vojnoviæ i Branka Vasiljeviæ<br />
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd<br />
kgmB gen iz proizvoðaèa nebramicinskog kompleksa antibiotika, Streptomyces<br />
tenebrarius, kodira KgmB metilazu koja obezbeðuje rezistenciju na kanamicin i<br />
gentamicin. KgmB metilaza modifikuje ciljno mesto na 16S rRNK i na taj naèin štiti<br />
proizvoðaèa od sopstvenog toksiènog produkta. Ekspresija kgmB gena je autoregulisana<br />
na nivou translacije. Regulatorni model je najpre testiran in vivo korišæenjem kgmB-lacZ<br />
genskih fuzija. U cilju prouèavanja ovog procesa in vitro, KgmB protein je eksprimiran u<br />
E. coli i preèišæen pomoæu QIAexpress sistema. Nedavno je pokazano da inaktivirani<br />
kgmB gen (inaktivacija u BglII restrikcionom mestu koja dovodi do sinteze skraæenog<br />
KgmB proteina sa samo 66 originalnih aminokiselina) smanjuje aktivnost kgmB-lacZ<br />
genske fuzije. Ovaj rezultat ukazuje da je N-terminalni region KgmB metilaze zaduen za<br />
regulaciju t.j. za vezivanje za iRNK. Da bi se ispitale osobine i funkcionalnost<br />
N-terminalnog regiona KgmB metilaze inaktivirani kgmB gen je kloniran u pQE30<br />
ekspresioni vektor. Eksprimirani protein obeleen histidinskim repiæem je preèišæen<br />
metal afinitetnom hromatografijom. Preèišæen skraæeni KgmB protein je analiziran<br />
elektroforetski i Western blotom. Funkcionalnost skraæenog KgmB proteina biæe ispitana<br />
eksperimentima RNK-gel šifta i uporeðena sa intaktnom KgmB metilazom.<br />
ANALYSIS OF TRUNCATED KgmB METHYLASE FROM S. tenebrarius<br />
The kgmB gene from Streptomyces tenebrarius, producer of nebramycin complex of antibiotics,<br />
encodes for kanamycin-gentamicin resistance methylase, KgmB. KgmB<br />
methylase modifies the target site on 16S rRNA and thus protect producer against its own<br />
toxic product. The kgmB gene is autogenously regulated at translational level. A regulatory<br />
model was first tested in vivo using kgmB-lacZ gene fusions. In order to study this<br />
process in vitro KgmB protein was overexpressed in E. coli and purified using<br />
QIAexpress System. Recent results showed that inactivated kgmB gene (inactivation in<br />
BglII restriction site which gives truncated protein with only 66 original amino acids) is<br />
able to repress activity of kgmB-lacZ gene fusions. This finding suggests that N-terminal<br />
region of KgmB methylase is responsible for regulation i.e. for binding to mRNA. With<br />
the intention of analyzing properties and functionality of N-terminal region of KgmB<br />
protein inactivated kgmB gene was cloned in pQE30 expression vector to make a construct<br />
that places the 6xHis tag at the N-terminus of the protein. For purification of<br />
6xHis-tagged protein metal affinity chromatography was used. Purified truncated KgmB<br />
protein was analyzed electrophoreticaly and on Western blot. Functionality of truncated<br />
KgmB protein would be examined by RNA-gel shift experiments and compared with intact<br />
KgmB methylase.