S. cerevisiae ELP1 to ELP6 genes were amplified in polymerase chain reactions (PCRs) and cloned in pairs into vectors with opposite-oriented, inducible GAL1 and GAL10 promoters to allow simultaneous expression of two genes from one vector (59). Each vector harbored different selection marker cassettes (LEU2, TRP1, and URA3) to ensure the exclusive growth of transformants. For the purpose of later protein purification, the ELP3 or ELP1 gene was modified with FLAG and Twin-Strep-tag (IBA Lifesciences) coding sequences, while the C terminus of Elp6 was fused to Staphylococcus aureus protein A and a cleavage site for the protease of tobacco etch virus (His-TEV). Elp1, Elp2, and Elp3 mutant proteins were prepared by mutating respective genes in modified QuikChange reactions. DS1-2b yeast strain was cotransformed with vectors carrying ELP genes. Transformants were grown on selection medium agar plates. To overproduce Elongator, transformed cells were grown at 30°C with shaking in raffinose selection medium to an OD600 of 0.3 to 0.6 and induced for the following 21 hours with galactose at a 2% final concentration. Cells were next spun down, suspended in a lysis buffer [LB; 250 mM Hepes (pH 7.5), 100 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mM sodium orthovanadate, 20 mM sodium fluoride, aprotinin (2 μg/ml), leupeptin (5 μg/ml), 1 μM pepstatin A, and 1 mM PMSF; 3 ml of LB per 20 g of cell pellet], and frozen in liquid nitrogen in the form of small drops. Drops were lysed by cryo-milling to a powder on a Qiagen TissueLyser II instrument and stored at −80°C. All purification steps were performed at 4°C or on ice. To isolate recombinant wild-type and mutant forms of Elongator, 12 ml of LB was added per 24 to 26 g of yeast pellet and thawed for 1 hour with agitation. Lysates were clarified by centrifugation first at 7000g (30 min) followed by another round of centrifugation at 20,000g (20 min). Cell extracts were incubated with agitation for 30 min with 2 ml of immunoglobulin G Sepharose 6 Fast Flow affinity resin (GE Healthcare), followed by two wash steps (10 ml). The bound fraction was incubated with 1 mg of His-TEV protease in the presence of 1 mM DTT for 1 hour and eluted. Next, Elongator was bound to a 1-ml StrepTrap HP column (GE Healthcare) and eluted with 20 mM d-desthiobiotin. After concentrating on an Amicon Ultra-15 100k, Elongator was purified on a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with 20 mM Hepes (pH 7.5) buffer, 100 mM NaCl, and 3 mM DTT. Selected fractions were pooled and concentrated to ~1 mg/ml with an Amicon Ultra-0.5 100k concentrator. Aliquots were frozen in liquid nitrogen and stored at −80°C.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.