DIVERSE System: De Novo Creation of Peptide Tags for Non-enzymatic Covalent Labeling by In Vitro Evolution for Protein Imaging Inside Living Cells  Takashi Kawakami, Koji Ogawa, Naoki Goshima, Tohru Natsume  Chemistry & Biology  Volume 22, Issue 12, Pages 1671-1679 (December 2015) DOI: 10.1016/j.chembiol.2015.10.016 Copyright © 2015 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2015 22, 1671-1679DOI: (10. 1016/j. chembiol. 2015 Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Schematic Representation of the DIVERSE System for the Development of Protein-Labeling Peptide Tags A DNA library containing randomized sequences is transcribed and spontaneously modified with puromycin linkers inside the cell-free coupled transcription/translation system. The puromycin-modified mRNA library is translated into a peptide library; the expressed peptides are spontaneously displayed on their encoding mRNAs through a puromycin-DNA linker in the system. The non-covalently mRNA-displayed peptide library is then reverse-transcribed to form a covalent cDNA-displayed peptide library. The cDNA-displayed peptide library is precleared, without purification, with target-free beads for the removal of bead-binding peptide tags. The precleared peptide library, which contains DTT at several-millimolar concentrations, is then reacted with target-immobilized beads. The beads are stringently washed for the isolation of target-reactive peptide tags. The recovered cDNAs encoding the reactive peptide tags are amplified by PCR and used for the next round of selection. In the present study, the de novo creation of non-enzymatically covalent-labeling peptide tags was performed by selection from a random peptide library (fM-X8-15-G5S) using p-(chloromethyl)benzamide (pClBz), which is shown in red, as a small-molecule target. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Progress During the DIVERSE System-Based Selection of pClBz-Reactive Peptide Tags from a Random Peptide Library and the Selected Sequences (A) Progress during DIVERSE system-based selection from a random library (∼1014 in library size) prepared using a cell-free translation system. Recovery of cDNAs in each round of selection was determined by qPCR. The selection pressure was gradually increased by decreasing the reaction time from 60 min to 3 min. The DNA input into rounds 11, 12, and 13 was subjected to error-prone PCR amplification. (B) Sequences of the abundant peptides selected from the library. The two pClBz-reactive peptides (THWFWCPYWGWRLS and WFWCPYWRTYIWY) were named CRP1 and CRP2, respectively. The pClBz-reactive cysteine residue is highlighted in red. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Site Specificity of Polypeptide Labeling Using pClBz-Reactive Peptide Tags Evolved by the DIVERSE System (A) Reaction between p-(chloromethyl)-N-(2-hydroxyethyl)benzamide and CRP-fused model peptide in the in vitro translation system. (B) MALDI-TOF MS spectra of CRP-fused peptides prepared using the in vitro translation system (left) and those reacted with p-(chloromethyl)-N-(2-hydroxyethyl)benzamide (right) for CRP1 tags (upper) and CRP2 tags (lower). Calculated (Calc.) and observed (Obsd.) molecular masses for singly charged species [M + H]+ of desired peptides are shown in each spectrum. Minor peaks (∗) are unknown expression products that existed before pClBz labeling. (C) Reaction between p-(chloromethyl)-N-(2-hydroxyethyl)benzamide and CRP-fused peptides containing an additional cysteine residue in the in vitro translation system. (D) MALDI-TOF MS spectra of CRP-fused peptides containing an additional cysteine residue prepared using the in vitro translation system (left) and those reacted with p-(chloromethyl)-N-(2-hydroxyethyl)benzamide (right) for CRP1 tags (upper) and CRP2 tags (lower). Calculated (Calc.) and observed (Obsd.) molecular mass for the singly charged species [M + H]+ of the desired peptides are shown in each spectrum. Minor peaks (∗) are unknown expression products that existed before pClBz labeling. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Specificity of Protein Labeling Using pClBz-Reactive Peptide Tags Evolved by the DIVERSE System (A) Reaction between fluorescein-modified pClBz and CRP-fused DHFR in the in vitro translation system. (B) SDS-PAGE analysis of reaction products between fluorescein-modified pClBz and CRP-fused DHFR prepared using the in vitro translation system. In vitro translation products labeled with fluorescein-modified pClBz were resolved by SDS-PAGE and visualized by Coomassie brilliant blue stain (left) or in-gel fluorescence (right). Lanes 2 and 3 show fluorescein labeling of expressed DHFR fused with CRP1 and CRP2 tags (22 kDa), respectively. Negative control, DHFR expressed without a tag, is shown (lane 1). MK indicates protein marker. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 5 Generality of Protein Labeling Using pClBz-Reactive Peptide Tags Evolved by the DIVERSE System (A) Labeling of C-terminal CRP-fusion proteins. SDS-PAGE analysis of the products of the reaction between fluorescein-modified pClBz and C-terminal CRP-fusion DHFR prepared using the in vitro translation system is shown. In vitro translation products labeled with fluorescein-modified pClBz were resolved by SDS-PAGE and visualized by fluorescence. Lanes 1 and 2 show fluorescence labeling of DHFR fused with the CRP1 and CRP2 tag, respectively. (B) Labeling of other CRP1-fusion proteins. SDS-PAGE analysis of the products of the reaction between fluorescein-modified pClBz and CRP1-fused proteins prepared using the in vitro translation system is shown. In vitro translation products labeled with fluorescein-modified pClBz were resolved by SDS-PAGE and visualized by fluorescence. Lanes 2 and 3 show fluorescence labeling of tumor necrosis factor-α (TNF, 29 kDa) and peptidyl-prolyl cis-trans isomerase (PPIA, 21 kDa) fused with the CRP1 tag, respectively. (C and D) Protein labeling with other fluorescent probe-modified pClBz. (C) Chemical structure of pClBz modified with the fluorescent probes coumarin, Alexa Fluor 488 (AF488), and tetramethylrhodamine (TMR). (D) SDS-PAGE analysis of the products of the reaction of fluorescent probe-modified pClBz and a CRP1-DHFR fusion protein prepared using the in vitro translation system. In vitro translation products labeled with fluorescent probe-modified pClBz were resolved by SDS-PAGE and visualized by fluorescence. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 6 Irreversibility of the pClBz Labeling of a CRP-Fused Protein (A) Treatment of fluorescein-pClBz-labeled CRP-tagged DHFR and FlAsH-labeled Cys4-tagged DHFR with 2-mercaptoethanol at 95°C for 5 min. (B) SDS-PAGE analysis of fluorescein-pClBz-labeled CRP1-DHFR and FlAsH-labeled Cys4-DHFR. In vitro translation products labeled with fluorescein-modified pClBz or FlAsH were incubated with 2-mercaptoethanol (BME), resolved using SDS-PAGE, and visualized by fluorescence. Lanes 1 and 2 show fluorescein-pClBz-labeled CRP1-DHFR with and without BME treatment, respectively. Lanes 3 and 4 show FlAsH-labeled Cys4-DHFR with and without BME treatment, respectively. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 7 Intracellular Labeling of CRP Peptide Tag Fusion Proteins (A) HEK cells transiently expressing CRP-fused GFP targeted to the nucleus (CRP-GFP-NLS) were labeled with 1 μM TMR-pClBz at 37°C for 30 min. The nuclear localization sequence was (DPKKKRKV)3. (B) Representative microscopic images of HEK cells expressing CRP-GFP-NLS fusions labeled with TMR-pClBz at 30 min after washing three times with media. The first column shows the GFP images, the second column shows the TMR-pClBz, the third column shows the bright-field images merged with the GFP images, and the fourth column shows the TMR images merged with the GFP images. The images were taken at 40× magnification. Chemistry & Biology 2015 22, 1671-1679DOI: (10.1016/j.chembiol.2015.10.016) Copyright © 2015 Elsevier Ltd Terms and Conditions