Prepared DNA templates. ProX tag sequence (SKQIEVN–amber-SNE) was contained in PEGx(ProX)–FLAG.

<p>Prepared DNA templates. ProX tag sequence (SKQIEVN–amber-SNE) was contained in PEGx(ProX)–FLAG.</p

Illustration of the synthesis of polyethylene-glycol-carrying tRNA and the incorporation of PEG into a polypeptide by in vitro translation.

<p>Illustration of the synthesis of polyethylene-glycol-carrying tRNA and the incorporation of PEG into a polypeptide by in vitro translation.</p

Western blot analysis using anti-FLAG tag antibody of the in vitro translation of thioredoxin (Trx) with an incorporated PEG chain.

<p>a. Dependence of protein synthesis on PEG–AF–tRNA concentration. b. PEG-incorporated protein synthesis under different conditions.</p

Mass spectra of polyethylene-glycol-carrying tRNAs.

<p>The molecular weights of PEG16–tRNA, PEG24–tRNA, and PEG24–tRNA were calculated to be 24485.69, 24838.10, and 25983.45, respectively.</p

Mass spectrometry analysis of poly(ethylene glycol)-incorporated polypeptide.

<p>a. Mass spectra of polypeptide incorporated with poly(ethylene glycol) chain. PEG4–<i>FLAG</i>, calculated 2681.177 for (M+H)⁺, found 2681.152; PEG12–<i>FLAG</i>, calculated 3033.386 for (M+H)⁺, found 3033.972; PEG16–<i>FLAG</i>, calculated 3209.492 for (M+H)⁺, found 3209.529; PEG24–<i>FLAG</i>, calculated 3563.890 for (M+H)⁺, found 3563.712; PEG48–<i>FLAG</i>, calculated 4709.244 for (M+H)⁺, not found. b. Molecular weight dependence of coefficient of incorporation of poly(ethylene glycol) into a polypeptide by in vitro translation.</p

Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group

To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-<i>N</i>,<i>N</i>-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity