Protein Engineering Protocols - Mycobacteriology research center

Ribosome-Inactivation Display System 231(29,30). To construct pStAv-mR and pGST-mR, the gene for RTA in each plasmid(pStAV-R or pGST-R) was replaced by a mutant gene for RTA by standardprocedures (31) with the following primers: 5′-TTG CAT CCA AAT GAT TTCACA AGC AGC ACA CTT CCA ATA TAT TAG GGA GAA ATG-3′ (underliningindicates mutations E177A and R180H) and 5′-GAT CCT AGC GTA ATT ACACTT GAC GAT CCT AGC GTA ATT ACA CTT GA-3′ (underlining indicatesmutation E208D).3.2. The Cycle of RIDS for the Selection of Functional ProteinDigest the modified pLRS containing the sequence of the random library orcDNA library (in our model study, pStAv-R, pGST-R, pStAv-mR, and pGST-mR)in the 3′-terminal region of the ORF by XhoI to yield linear DNA by standardrecombinant DNA methods. The digestion is indispensable for the termination ofthe transcription.3.2.1. Transcription of the DNA Library to mRNADNAs should be transcribed by T7 RNA polymerase with a cap-structureanalog. In our model study, DNAs were transcribed by a T7 Ampliscribe kit,according to the supplier’s recommendation (Epicentre Technologies Co.).1. Add 1 µg of linear DNA to 20 µL of the transcription mixture including a 3 mMcap-structure analog, 7.5 mM rATP, rCTP, and rUTP; 0.75 mM rGTP; and 10 mMdithiothreitol, reaction buffer, and AmpliScribe T7 Enzyme Solution, according tothe supplier’s recommendation.2. Incubate the reaction mixture for 2 to 4 h at 37°C to produce each mRNA (we callrespective mRNAs: StAv-R, pGST-R, pStAv-mR, and GST-mR).3. After the transcriptional reaction, digest the template DNA by DNase I completely,because residual template DNA influences the selection step.4. Purify mRNA with an RNeasy mini kit, according to the supplier’s recommendation(QIAGEN).3.2.2. Affinity Selection of the Ribosome–mRNA–Protein Complexand Isolation of mRNAPrepare the RNA-encoding library (in our model study, StAv-R, StAv-mR,GST-R, and GST-mR) and Flexi ® Rabbit Reticulocyte Lysate system (Promega;see Note 1).Fig. 3. (Continued) plasmid to construct pStAv-R and pGST-R. To confirm that RTAhelps to maintain a stable ribosome complex, in a control experiment, we made pStAv-mRand pGST-mR encoding mutant (inactive) RTA by site-specific mutagenesis. (B) DNAsequence of pStAv-R used for the RIDS constructs. (C) DNA sequence of pGST-R usedfor the RIDS constructs.