Journal: International Journal of Molecular Sciences

Article Title: Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides

doi: 10.3390/ijms242115661

Figure Lengend Snippet: ( A ) Ribosome display selection in combination with genetic code expansion for identifying the X-peptide of interest. (I) In vitro transcription of DNA constructs, including an amber stop codon (TAG) for generating an mRNA pool that encodes various peptides. (II) In vitro translation of the mRNA pool in the presence of 20 types of AA-tRNAs and synthetic X-tRNA UAG to form X-peptide-ribosome-mRNA complexes that can link the X-peptides as phenotypes and the corresponding mRNAs as genotypes. (III) In vitro selection of ribosomal complexes that display X-peptides interacting with monoclonal anti-peptide antibodies immobilized on beads (MPA-B). (IV) Recovery of mRNAs by disassembling the selected ribosomal complexes. (V) Reverse transcription (RT)-PCR for synthesis of DNAs used to perform the next round of selection or to identify X-peptides. ( B ) Electrophoretic gel analysis of RT-PCR products derived from the mRNAs that encode the sequences of UAG-FLAG peptides after the ribosome display selection against MFA-B. FI = fluorescent intensity, BS = before selection, 1R = the first round of selection, and 2R = the second round of selection. In the initial mRNA pool, the molar amount of mRNA of the UAG-FLAG peptide of interest was 1/10 5 times that of the V5 peptide as its counterpart. The mRNAs were recovered in each round of selection after in vitro translation in the presence (+) or absence (−) of Trp-tRNA UAG . The RT-PCR products were quantified on the basis of the fluorescent band intensities in the electrophoretic gel image.

Article Snippet: The resulting solutions were added to 15 µL of MFA-B (ANTI-FLAG M2 Affinity Gel, Merck, Darmstadt, Germany) or monoclonal anti-V5 peptide antibodies immobilized on beads which consisted of Anti-V5-tag mAb-Biotin (MBL, Tokyo, Japan) and High-Capacity NeutrAvidin Agarose (ThermoFisher Scientific).

Techniques: Selection, In Vitro, Construct, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides

doi: 10.3390/ijms242115661

Figure Lengend Snippet: ( A ) Western blot analysis of the expression levels of X-FLAG peptides (X = p Bzo-Phe, Tyr, and Trp), depending on the type of synthetic X-tRNA UAG . To generate X-FLAG peptides fused with a protein spacer (PS) and a hexa-histidine tag (6H), namely p Bzo-Phe-FLAG-PS-6H, Tyr-FLAG-PS-6H, and Trp-FLAG-PS-6H, in vitro translations of the mRNA encoding the sequence of UAG-FLAG-PS-6H were performed in the presence of p Bzo-Phe-tRNA UAG , Tyr-tRNA UAG , and Trp-tRNA UAG , respectively. ( B ) Schematic representation for analyzing photo-cross-linking reaction between p Bzo-Phe-FLAG peptide-fused protein ( p Bzo-Phe-FLAG-PS-6H) and MFA-B. (i) Interaction of p Bzo-Phe-FLAG-PS-6H with MFA. (ii) Photo-irradiation for activating p Bzo-Phe unit and inducing photo-cross-linking reaction. (iii) Addition of synthetic FLAG peptides (SFPs) for eluting p Bzo-Phe-FLAG-PS-6H that did not bind to MFA-B via a covalent bond. (iv) Addition of monoclonal anti-hexa-histidine tag antibody conjugated with a peroxidase (MHA-P) for detecting the fusion proteins bound covalently to MFA-B by chemiluminescence. ( C ) Bright-field and chemiluminescence images of MFA-B with or without photo-irradiation and addition of SFPs.

Article Snippet: The resulting solutions were added to 15 µL of MFA-B (ANTI-FLAG M2 Affinity Gel, Merck, Darmstadt, Germany) or monoclonal anti-V5 peptide antibodies immobilized on beads which consisted of Anti-V5-tag mAb-Biotin (MBL, Tokyo, Japan) and High-Capacity NeutrAvidin Agarose (ThermoFisher Scientific).

Techniques: Western Blot, Expressing, In Vitro, Sequencing, Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides

doi: 10.3390/ijms242115661

Figure Lengend Snippet: Electrophoretic gel analysis of RT-PCR products derived from the mRNAs that encode ( A ) UAG-FLAG(Y2A) peptide or ( B ) V5 peptide after ribosome display selection against MFA-B. FI = fluorescent intensity, BS = before selection, 1R = the first round of selection, 2R = the second round of selection, and 3R = the third round of selection. In the initial mRNA pool, the molar amount of mRNA of the UAG-FLAG(Y2A) peptide of interest was 1/10 5 times that of the V5 peptide as its counterpart. Then, p Bzo-Phe was inserted into the position of the amber stop codon through in vitro translation in the presence of p Bzo-Phe-tRNA UAG , leading to the formation of p Bzo-Phe-FLAG(Y2A) peptide-ribosome-mRNA complexes. The mRNAs were recovered in each round of selection after photo-irradiation (+) or non-irradiation (−), followed by the addition of synthetic FLAG peptides. ( C ) Augmentation of p Bzo-Phe-FLAG(Y2A) peptide (i) and reduction of V5 peptide (ii) after the third round of ribosome display selection. The RT-PCR products were quantified on the basis of the fluorescent band intensities in the electrophoretic gel image.

Article Snippet: The resulting solutions were added to 15 µL of MFA-B (ANTI-FLAG M2 Affinity Gel, Merck, Darmstadt, Germany) or monoclonal anti-V5 peptide antibodies immobilized on beads which consisted of Anti-V5-tag mAb-Biotin (MBL, Tokyo, Japan) and High-Capacity NeutrAvidin Agarose (ThermoFisher Scientific).

Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Selection, In Vitro, Irradiation

Journal: Cell reports

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer

doi: 10.1016/j.celrep.2018.08.089

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The mixtures were then subjected to HA or FLAG immunoprecipitation using 30 μL of pre-washed anti-HA agarose (Thermo Fisher Pierce) or anti-FLAG M2 affinity agarose (Sigma-Aldrich) beads, respectively, followed by western analysis using 1:1000 dilutions of HA (Sigma-Aldrich, #12CA5), FLAG (Sigma-Aldrich, #F7425), and BID (Santa Cruz, #sc-11423) antibodies.

Techniques: Virus, Recombinant, Synthesized, Protease Inhibitor, Staining, Bicinchoninic Acid Protein Assay, Mutagenesis, LDH Cytotoxicity Assay, Silver Staining, Membrane, Plasmid Preparation, Software