Zbornik - Društvo genetičara Srbije

70 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Usm-3
Subotica, 30. novembar - 4. decembar 2004.
PREÈIŠÆAVANJE SGM METILAZE IZOLOVANE IZ Micromonospora
zionensis I DETERMINACIJA SEKUNDARNE STRUKTURE
Tatjana Iliæ-Tomiæ, M. Saviæ i Branka Vasiljeviæ
Institut za molekularnu genetiku i genetsko inenjerstvo, Beograd
Iz Micromonospora zionensis, proizvoðaèa antibiotika G-52, kloniran je sgm gen koji
kodira metilazu koja modifikuje 16S rRNK u okviru male ribozomalne subjedinice i tako
štiti bakteriju od sopstvenog toksiènog proizvoda.
U cilju preèišæavanja Sgm proteina, sgm gen je ukloniran u Qiaexpress pQE vektor, pod
promotor-operatorski element T5 faga, koji se sastoji od promotora koga prepoznaje E.
coli RNK polimeraza i dve lac operatorske sekvence koje poveæavaju vezivanje lac
represora. Ovim konstruktom se repiæ od 6 histidina pozicionira na N terminus proteina.
Aktivnost His6-Sgm proteina je potvrðena in vivo.
Preèišæavanje His6-Sgm proteina je obavljeno na Ni-NTA koloni afinitivnom hromatografijom
pod nativnim uslovima i prisustvo proteina je detektovano na SDS poliakrilamidnom
gelu.
Snimljen je CD spektar (cirkularni dihroizam) preèišæenog Sgm proteina. Prema
podacima dobijenim iz CD spektra utvrðeno je da su á heliksi i zavojnice predominantni
elementi sekudarne strukture što je u saglasnosti sa kompijuterski zasnovanim predikcijama.
Sekundarna struktura Sgm proteina odreðena CD spektroskopijom pokazuje
visok stepen homologije sa â-laktamazom S. aureus iako na aminokiselinskom nivou
nema homologije.
PURIFICATION OF THE SGM METHYLASE ISOLATED FROM
Micromonospora zionensis AND DETERMINATION OF THE SECONDARY
STRUCTURE
The sgm gene, coding for specific 16S rRNA methylase that modifies the target site on
small ribosomal subunit and thus protects the producer against its own toxic product, was
cloned from Micromospora zionensis, producer of antibiotic G52.
In order to purify the Sgm protein, the sgm gene was cloned under optimized promoter-operator
element consisting of phage T5 promoter (recognized by the E. coli RNA
polymerase) and two lac operator sequences which increase lac repressor binding, in
QIAexpress pQE vectors. This construct places the 6xHis tag at the N-terminus of the
protein. The functional activity of His6-Sgm fusion protein was confirmed in vivo. Purification
of His6-tagged protein by Ni-NTA affinity chromatography was carried out under
native conditions and the protein was detected on sodium dodecyl sulphate
polyacrylamide gel.
Purified protein was used in circular dichroism (CD) spectroscopy studies. According to
CD spectral data protein was mostly helically structured with a lot of amino acid residues
placed in turns connecting helices that is in agreement with results based on computer
secondary structure prediction. The closest mach in secondary structure database
was -lactamase from S. aureus. Protein sequences alignment showed no significant sequence
homology between this two proteins.