Zbornik - Društvo genetičara Srbije

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