Protein Engineering Protocols - Mycobacteriology research center

232 Fujita et al.1. Add 2 µg mRNA to 40 µL of the translation mixture including 33 µL of Flexi ®Rabbit Reticulocyte Lysate, 40 mM KCl, 40 µM total amino acid mixture, and 40 URecombinant RNasin ® Ribonuclease Inhibitor (Promega), and adjust the volumeof the solution to 50 µL with distilled water. Do not add dithiothreitol and Mg 2+ion (see Note 1).2. Incubate for 20 min at 30°C. In our study, three sets of mRNA were translated(2 µg of each set: a mixture of mRNAs in the ratio of 1:1 or each mRNA encodingstreptavidin or GST [StAv-R and GST-R] as a control; see Note 2).3. After translation, add 1 mL of the appropriate binding buffer (see Heading 2.,item 13) and ligand-immobilized beads. In our study, we add 10 µL of a suspensionof biotin–agarose or glutathione beads (see Note 3).4. Incubate for 1 h at 4°C with gentle rotation for the binding reaction. Although theincubation can be performed at room temperature, we recommend performing thereaction at 4°C, because nonspecific binding was sometimes detected in our studyat reverse transcriptase (RT)-PCR (see Note 4).5. After the binding reaction, centrifuge the mixture for 1 min at 500g to precipitatethe ligand-immobilized beads.6. Remove supernatant containing unbound mRNA and protein.7. Add 200 µL of washing buffer (see Subheading 2., item 13) and mix gently for30 s.8. Centrifuge the mixture for 1 min at 500g to precipitate ligand-immobilized beads.9. Remove supernatant containing unbound mRNA and protein.10. Repeat the wash steps 7 to 9 two to three times.11. Add 100 µL of elution buffer (see Subheading 2, item 16) and shake vigorously atroom temperature for 30 min to isolate the bound mRNA from ligand-immobilizedbeads.12. Purify eluted mRNA with the RNeasy kit, according to the supplier’s recommendation,and concentrate purified mRNA by vacuum pump.3.2.3. Reverse TranscriptionPerform the reverse transcription reaction using RT. In our model study, thereverse transcription reaction was performed using ReverTra Ace ® (Toyobo),according to the supplier’s recommendation.1. Mix the purified mRNA, 4 µL of 10 µM RT primer, 2 µL of each 10 mM dNTP,and 4 µL of 5X RT buffer. Adjust the volume of the solution to 18 µL with distilledwater, without adding RNase inhibitor and ReverTra Ace, according to thesupplier’s recommendation. An RT primer (5′-GTG TAG CTT TGC ACA GTGGC-3′) that recognizes the upstream portion of the RTA sequence was used.2. Denature the mixture at 65°C for 5 min and chill on ice immediately.3. Add 1 µL of RNase inhibitor and 1 µL of ReverTra Ace to the mixture.4. Incubate at 42°C for 1 h for the reverse transcription reaction. To inactivate theenzyme, incubate at 99°C for 5 min.