XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta
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Posters<br />
86.<br />
IN VIVO CROSS-LINKING FOR IDENTIFICATION OF TELLURITE<br />
RESISTANCE-ASSOCIATED PROTEINS<br />
Jana Schubertová Aradská 1 , Dušan Blaškovič 1 and Ján Turňa 1,2<br />
1<br />
Department of molecular biology PriF UK Bratislava, 2 Department of molecular<br />
biology SAV Bratislava<br />
In spite of the fact that a number of tellurite-resistance determinants have been<br />
characterized, little is known about the mechanism responsible for this resistance.<br />
Determinant encoding resistance against pottasium tellurite was discovered in clinical<br />
isolate of Escherichia coli KL53 on a large conjugative plasmid pTE53. Analyses showed<br />
that genes terB, terC, terD and terE are essential for conservation of the resistance. In<br />
order to understand this mechanism we are looking for tellurite resistance associated<br />
proteins. For initial investigation of protein-protein interactions we decided to use in vivo<br />
chemical cross-linkings to effectively capture and identify interacting proteins. Chemical<br />
cross-linking stabilizes interactions through covalent bond formation, allowing the detection<br />
of protein-protein interactions in native cells that are weak and/or transient. The<br />
most commonly used cross-linking reagent is formaldehyde, a water-soluble and cell<br />
membrane-permeable molecule, which provides fast and reversible cross-linking reaction.<br />
With this in mind, we decided to investigate the effects of formaldehyde cross-linking.<br />
All four genes were cloned to pET expression system to produce His-tagged proteins<br />
and constucts were transformed to E. coli BL21(DE3). These strains were subequently<br />
co-transformed with plasmid pLK18 which contains the functional part of the tellurite<br />
resistance operon, encompasing terBCDEF. Strains were cultivated on medium with tellurite<br />
to induce oxidative stress and living cells were treated with formaldehyde. Crosslinked<br />
TerB, TerC, TerD and TerE proteins were purificated by Ni-NTA resin and analyzed by<br />
SDS-PAGE. In the next step we would like to identify associated proteins by MS analyses.<br />
<strong>XXII</strong>. Biochemistry Congress, Martin<br />
209
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