Fig 1.
Determination of minimum epitope sequence of Ra62 antibody.
(A,B) Narrowing epitope region of Ra62 antibody by AlphaScreen. Biotin-SrtA-DRD1 C-terminal fragments were cell-free synthesized. Interactions between DRD1 fragments and Ra62 or Ra48 antibodies were evaluated by AlphaScreen (panel A) and Western blotting (panel B). Error bars in panel A indicate standard deviation (n = 4). Synthesis of biotinylated fusion protein was visualized by using anti-biotin antibody (panel B). (C) Ra62 antibody recognized 5 residues of DRD1 at its C-terminus. C-terminal DRD1 7-residue fragment was fused with Venus protein [26], and alanine mutation and deletion were introduced into it. Mutant proteins were synthesized by wheat cell-free system, and subjected to Western blotting with Ra62 antibody and anti-rabbit IgG-HRP as primary antibody. Synthesis of Venus protein was visualized by using anti-GFP antibody. (D) Ra62 antibody recognized terminus carboxyl group in the epitope peptide. Interaction between antibody and biotinylated peptide was detected using AlphaScreen. (E) Specificity of Ra62 antibody. Extracts from wheat germ or cultured cells were subjected to Western blotting using Ra62 antibody as primary antibody. Cell-free synthesized DRD1 was applied as positive control recognized by Ra62 antibody. CF, reaction mixture of cell-free synthesis containing wheat germ extract.
Fig 2.
(A) Kinetics assay of D1CE epitope and Ra62 antibody. Ra62 antibody was captured on a protein G-immobilized sensorchip at 200 RU. Purified FLAG-GST-D1CE protein was then injected for 120 sec as analyte. Black lines represent a global fit of a 1:1 interaction model to each kinetic data set. (B) Elution scouting. Ra62 antibody was covalently immobilized on a sensorchip. In each cycle, FLAG-GST-DRD1 (337–446) or FLAG-GST-DRD1 (337–446, P445V) was injected and then an eluate was injected. Sensorgram of elution scouting is shown in S2 Fig. Dissociation rate is calculated by dividing binding response at 160 sec after eluate injection (S2 Fig, filled arrowhead) by one at 10 sec before eluate injection (S2 Fig, blank arrowhead). (C) D1CE mutants. Three residues on the C-terminus of FLAG-GST-DRD1 (337–446) were substituted one by one by similar amino acids. Their expression (anti-FLAG, lower panel) and Ra62 antibody reactivity (upper panel) were examined by Western blotting. (D) Binding assay of D1CE mutants. FLAG-GST-DRD1 (337–446) and mutants were injected to Ra62 antibody-immobilized sensorchip. Black box shows dissociation curves, in which difference in the dissociation rate was emphasized by stretching Y axis and shrinking X axis. (E) Kinetics assay of CP5 tag. Ra62 antibody was captured on a protein G-immobilized sensorchip at 200 RU. Purified FLAG-GST-CP5 protein was injected to Ra62 captured sensorchip for 120 sec as analyte. (F) Purification of CP5 tagged protein using CP5 system. Cell-free synthesis reaction mixture of FLAG-GST-DRD1 (337–446, P445V) protein was mixed with Ra62 antibody Sepharose. After washing, captured protein was eluted by D1CE epitope peptide. Purified protein was visualized by SDS-PAGE and CBB staining. Arrowhead indicates position of FLAG-GST-DRD1 (337–446, P445V).
Fig 3.
Purification of soluble proteins by CP5 system.
(A) Purification of CYLD-CP5. Cell-free synthesized CYLD-CP5 was purified by CP5 system. Each fraction was subjected to SDS-PAGE and CBB staining. (B) Purification of control Venus-CP5 by CP5 system. (C) DUB assay. Linear tetra ubiquitin chain was treated with purified CYLD-CP5 and Venus-CP5. Degradation of products was visualized by SDS-PAGE and Ruby staining. (D) Purification of cell-free synthesized MARCH3-CP5 by CP5 system. Each fraction was subjected to SDS-PAGE and CBB staining. (E) Self-ubiquitination assay of purified MARCH3-CP5. Purified MARCH3-CP5 was mixed with E1, E2 (UbcH6), and HA-ubiquitin. Reaction mixture was applied to Western blotting. Ubiquitination was detected by HRP conjugated anti-HA antibody. (F) Recovery rate of soluble proteins purified by CP5 system. Signal intensity of each band in CBB staining images was measured by using ImageJ software. After background noise level was subtracted, signal intensity of CP5 tagged protein was plotted. Figures on the bar graph indicate recovery rate calculated by dividing recovered target proteins (elution 1 to 3) by total captured proteins (elution 1 to 3 and resin).
Fig 4.
Purification of GPCRs by CP5 tag.
(A) Purification of DRD1 P445V. Cell-free synthesized DRD1 P445V proteoliposome was solubilized by DDM, and was applied to CP5 system. Each fraction was analyzed by SDS-PAGE and CBB staining. C, crude; FT, flow through; W1-3, wash 1 to 3; E1-3, elution 1 to 3; R, resin. (B) Recovery rate of GPCRs purified by CP5 system. Figures on the bar graph indicate recovery rate calculated by dividing recovered target proteins (elution 1 to 3) by total captured proteins (elution 1 to 3 and resin). (C) Purification of CHRM2-CP5 expressed by baculovirus-insect cell system. CP5 tagged CHRM2 was expressed in SF9 cells. Membrane fraction was isolated and solubilized by DDM, and solubilized CHRM2-CP5 was purified by CP5 system. Each fraction was applied to SDS-PAGE and CBB staining. M, cell membrane fraction; S, solubilized fraction; FT, flow through; E1-9, elution 1 to 9; R, resin.