recombinant mbp tag protein  (Proteintech)

Journal: The Journal of Biological Chemistry

Article Title: The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

doi: 10.1074/jbc.M117.776021

Figure Lengend Snippet: KH3 domain of PCBP2 was most important for the HO1/PCBP2 interaction. A, schematic of the PCBP2 constructs. The full-length of PCBP2 (FL) consists of 366 amino acids. ΔKH1, ΔKH2, ΔKH3, and ΔLinker indicate the deletion mutants that lack 12–80 amino acids, 94–192 amino acids, 285–357 amino acids, and 193–284 amino acids, respectively. B, analysis of the interaction between DMT1 and PCBP2 deletion mutants by using a co-immunoprecipitation assay. (i) DMT1-GFP stably expressing HEp-2 cells were transfected with the N-terminal FLAG-tagged full-length form of PCBP2 and the deletion mutants of PCBP2. Notably, the 3× FLAG tag contains 39 amino acids. After 48 h of incubation, cells were lysed in TNE buffer. Then 5 μg of the total cell lysates were analyzed by Western blotting (WB) using anti-GFP pAb, anti-FLAG mAb, or anti-α-tubulin mAb. A total of 60 μg of these samples were co-immunoprecipitated (IP) with anti-GFP pAb-conjugated protein A beads and analyzed by Western blotting using anti-FLAG mAb. (ii) The results are typical of at least three independent experiments. The results shown in the bar graph are presented as the mean ± S.D. of three independent experiments. *, p < 0.05; ***, p < 0.001. C, analysis of the interaction between HO1 and PCBP2 deletion mutants by using a co-immunoprecipitation assay. (i) HO1-GFP stably expressing HEp-2 cells were transfected with the N-terminal FLAG-tagged full-length form of PCBP2 and the deletion mutants of PCBP2. After 48 h of incubation, cells were lysed in lysis buffer. Protein (5 μg) from total cell lysates was analyzed by Western blotting using anti-GFP pAb, anti-FLAG mAb, or anti-α-tubulin mAb. Then 60 μg of protein from these samples was co-immunoprecipitated with anti-GFP pAb-conjugated protein A beads and analyzed by Western blotting using anti-FLAG mAb. (ii) The results are typical of at least three independent experiments. Data are shown in the bar graph as the mean ± S.D. of three independent experiments. ***, p < 0.001. D, analysis of the interaction between HO1 and PCBP2 deletion mutants by using a pulldown assay. (i) MBP or MBP-HO1 was mixed with amylose resin for 30 min at room temperature and washed with p-lysis buffer. The resin-bound MBP or MBP-HO1 was eluted with 20 mm maltose/p-lysis buffer and analyzed by SDS-PAGE and stained with Coomassie R-250. (ii) pGEX2T or pGEX2T-PCBP2-FL, -ΔKH1, -ΔKH2, -ΔKH3, and -ΔLinker were expressed in E. coli and lysed in p-lysis buffer by sonication. The crude extracts were analyzed by SDS-PAGE and stained with Coomassie R-250. Arrowheads indicate the recombinant protein of interest. (iii) The crude extract from ii was mixed with the MBP or MBP-HO1-bound resin and incubated at 4 °C overnight. The resin was washed with p-lysis buffer three times. The binding proteins were eluted with 20 mm maltose/p-lysis buffer. MBP or MBP-HO1, which was bound to amylose resin or the crude extract that contained GST or GST-PCBP2, was analyzed by SDS-PAGE and stained with Coomassie R-250. The eluates were analyzed by Western blotting with anti-GST mAb. (iv) The results are typical of at least three independent experiments. The results shown in the bar graph are presented as the mean ± S.D. of three independent experiments. **, p < 0.01; ***, p < 0.001.

Article Snippet: Purification of recombinant proteins MBP-tagged proteins were purified using amylose resin (New England Biolabs).

Techniques: Construct, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Transfection, FLAG-tag, Incubation, Western Blot, Immunoprecipitation, Lysis, SDS Page, Staining, Sonication, Recombinant, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

doi: 10.1074/jbc.M117.776021

Figure Lengend Snippet: PCBP2 binds rapidly to a single class of non-interacting saturable binding sites on HO1 in vitro. A, time course of the binding of PCBP2 to HO1. (i and ii) The purified MBP-HO1 (10 μm) was mixed with amylose resin for 30 min at room temperature and washed with p-lysis buffer. Then, the purified GST-PCBP2 (10 μm) was loaded to MBP-HO1 (10 μm)/amylose resin and incubated at 37 °C for 0, 15, 30, 45, 60, 90, 120, 150, 180, or 300 s. MBP-HO1/amylose resin was washed with p-lysis buffer four times and eluted with 20 mm maltose/p-lysis buffer. MBP-HO1, bound to amylose resin was analyzed by SDS-PAGE and stained with Coomassie R-250. GST-PCBP2, which was used in this procedure, was analyzed by Western blotting using anti-GST mAb. The eluates were analyzed by Western blotting using anti-GST mAb. The results are typical of at least three independent experiments. The results shown in the line graph are presented as the mean ± S.D. of at least three independent experiments. B, PCBP2 demonstrates saturable binding to HO1. (i and ii) The purified MBP-HO1 (20 μm) was mixed with amylose resin for 30 min at room temperature and washed with p-lysis buffer. Then 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, and 20.0 μm purified GST-PCBP2 was loaded to MBP-HO1/amylose resin and incubated at 37 °C for 5 min. MBP-HO1/amylose resin was washed with p-lysis buffer four times and eluted with 20 mm maltose/p-lysis buffer. MBP-HO1, which was bound to amylose resin, was analyzed by SDS-PAGE and stained with Coomassie R-250. GST-PCBP2, which was used in this procedure, was analyzed by Western blotting using anti-GST mAb. The eluates were analyzed by Western blotting using anti-GST mAb. The results are typical of at least three independent experiments. The results shown in the line graph are presented as the mean ± S.D. of three independent experiments.

Article Snippet: Purification of recombinant proteins MBP-tagged proteins were purified using amylose resin (New England Biolabs).

Techniques: Binding Assay, In Vitro, Purification, Lysis, Incubation, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

doi: 10.1074/jbc.M117.776021

Figure Lengend Snippet: PCBP2 competes with CPR for binding to HO1 in vitro. A, expression of recombinant proteins. pGEX2T alone (GST; negative control), pET32 alone (His; negative control), pGEX2T-PCBP2, or pET32-CPRΔTM was expressed in E. coli and lysed in p-lysis buffer by sonication. The recombinant proteins were purified from E. coli cell extracts by GSH 4B beads or Ni-NTA beads and then analyzed by SDS-PAGE The results shown in the gel are typical of three independent experiments. B, competitive effect of His-CPR on the HO1/PCBP2 interaction. These studies were conducted in three stages. In the first step, the purified MBP or MBP-HO1 (10 μm) was mixed with amylose resin and then washed with p-lysis buffer (first reaction). In the second step, 10 μm purified pET32 alone (His), pGEX2T-PCBP2 (GST-PCBP2), or pET32-CPRΔTM (His-CPR) was mixed with the post-first reaction resin for 8 h at 4 °C and washed with p-lysis buffer (second reaction). In the third step, 10 μm purified GST-PCBP2, His, or His-CPR was mixed with the post-second reaction resin for 8 h at 4 °C and washed with p-lysis buffer (third reaction). The amylose resin-bound proteins were eluted with 20 mm maltose/p-lysis buffer. Eluates were analyzed by SDS-PAGE and stained with Coomassie R-250 staining or Western blotting (WB) using anti-GST mAb. MBP and MBP-HO1 were detected at 55 and 70 kDa in eluates, respectively. Results shown in the gels or blots are typical of three independent experiments. Data shown in the bar graph are presented as the mean ± S.D. of three independent experiments. ***, p < 0.001. C, competitive effect of GST-PCBP2 on the HO1/CPR interaction. These experiments were conducted in three stages: (1) MBP or MBP-HO1 (10 μm) was mixed with amylose resin and then washed with p-lysis buffer (first reaction); (2) 10 μm purified pGEX2T alone (GST), pET32-CPRΔTM (His-CPR), or pGEX2T-PCBP2 (GST-PCBP2) were mixed with the post-first reaction resin for 8 h at 4 °C and washed with p-lysis buffer (second reaction); and (3) then 10 μm purified His-CPR, GST, or GST-PCBP2 was mixed with the post-second reaction resin for 8 h at 4 °C and washed with p-lysis buffer (third reaction). The resin-bound proteins were eluted with 20 mm maltose/p-lysis buffer. Eluates were analyzed by SDS-PAGE and stained with Coomassie R-250 staining or Western blotting using anti-His mAb. MBP and MBP-HO1 were detected at 55 and 70 kDa in eluates, respectively. Results shown in the gels or blots are typical of three independent experiments. The results shown in the bar graph are presented as the mean ± S.D. of three independent experiments. ***, p < 0.001.

Article Snippet: Purification of recombinant proteins MBP-tagged proteins were purified using amylose resin (New England Biolabs).

Techniques: Binding Assay, In Vitro, Expressing, Recombinant, Negative Control, Lysis, Sonication, Purification, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

doi: 10.1074/jbc.M117.776021

Figure Lengend Snippet: Iron-loaded PCBP2 cannot bind to HO1. A, heme-loaded HO1 can bind to PCBP2. (i) Purified MBP-HO1 (10 μm) was mixed with amylose resin for 30 min at room temperature and incubated with 2.5, 5.0, or 10.0 μm heme for 10 min at 37 °C. The resin was washed with p-lysis buffer and mixed with 2.5 μm purified GST-PCBP2 for 5 min at 37 °C. The resin was washed with p-lysis buffer, and protein was eluted with 20 mm maltose in p-lysis buffer. MBP-HO1 bound to amylose resin was analyzed by SDS-PAGE and stained with Coomassie R-250. The eluates were analyzed by Western blotting (WB) with anti-GST mAb. The results are typical of at least three independent experiments. (ii) To confirm the formation of the holo-HO1 complex, the UV-visible absorbance was measured. After mixing heme with the MBP-HO1/amylose resin, the mixture was centrifuged and the supernatant collected. The absorbance was measured by using a 1:10 dilution of the sample. B, decrease in the interaction of PCBP2 with HO1 after loading PCBP2 with iron. (i) The purified MBP-HO1 (10 μm) was mixed with amylose resin for 30 min at room temperature. Then the purified GST-PCBP2 (2.5 μm) was preincubated with either the chelator, CDTA (10 μm; to deplete PCBP2 of iron), or freshly prepared solutions of 1, 2, 4, or 8 μm FeCl2 (to load PCBP2 with iron) for 10 min at 37 °C. MBP-HO1/amylose resin was loaded with iron-loaded GST-PCBP2 and incubated for 5 min at 37 °C. The resin was washed with p-lysis buffer three times. The protein was eluted with 20 mm maltose/p-lysis buffer. MBP-HO1, which was bound to amylose resin, was analyzed by SDS-PAGE and stained with Coomassie R-250. The eluates were analyzed by Western blotting (WB) with anti-GST mAb. The results are typical of at least three independent experiments. (ii) The results shown in the bar graph are presented as the mean ± S.D. of three independent experiments. ***, p < 0.001.

Article Snippet: Purification of recombinant proteins MBP-tagged proteins were purified using amylose resin (New England Biolabs).

Techniques: Purification, Incubation, Lysis, SDS Page, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The iron chaperone poly(rC)-binding protein 2 forms a metabolon with the heme oxygenase 1/cytochrome P450 reductase complex for heme catabolism and iron transfer

doi: 10.1074/jbc.M117.776021

Figure Lengend Snippet: Reconstruction of the HO1/PCBP2/CPR metabolon in vitro. A, recombinant GST-HO1–His-CPR complex can degrade heme into biliverdin. This study was performed in three steps. In the first step, the purified MBP-HO1 was mixed with amylose resin and then washed with p-lysis buffer (first reaction). In the second step, the purified pGEX2T-PCBP2 (GST-PCBP2) was mixed with the post-first reaction resin for 30 min at room temperature and washed with p-lysis buffer (second reaction). In the third step, the purified His-CPR was mixed with the post-second reaction resin for 30 min at room temperature and washed with p-lysis buffer (third reaction). During the third step, 5 μm heme and 25 μm NADPH were loaded to the mixture. The mixtures were then diluted 1:10, and absorbance was measured. The inset shows an enlargement of the spectrum between 650 and 720 nm that demonstrates the absorbance maximum of biliverdin at 680 nm (83). Results shown are typical of three experiments. B, HO1 alters its binding partner during heme degradation in vitro. In the first step, the purified MBP-HO1 was mixed with amylose resin and then washed with p-lysis buffer (first reaction). In the second step, the purified pGEX2T-PCBP2 (GST-PCBP2) or pET32-CPRΔTM (His-CPR) was mixed with the post-first reaction resin for 30 min at room temperature and washed with p-lysis buffer (second reaction). In the third step, the purified GST-PCBP2 or His-CPR was mixed with the post-second reaction resin for 30 min at room temperature and washed with p-lysis buffer (third reaction). During the third step, 5 μm heme alone or 5 μm heme and 25 μm NADPH were loaded to the mixture. The amylose resin-bound proteins were eluted with 20 mm maltose/p-lysis buffer. Eluates were analyzed by Western blotting (WB) using anti-GST mAb or anti-His mAb. The results are presented as the mean ± S.D. of three independent experiments. **, p < 0.01; ***, p < 0.001.

Article Snippet: Purification of recombinant proteins MBP-tagged proteins were purified using amylose resin (New England Biolabs).

Techniques: In Vitro, Recombinant, Purification, Lysis, Binding Assay, Western Blot

Journal: PLoS ONE

Article Title: The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization, Stress Granules, and Detergent-Insoluble Aggregates of FUS/TLS

doi: 10.1371/journal.pone.0049267

Figure Lengend Snippet: (A) in vitro methylation assay. Bacterial expressed GST-FUS or GST alone (control) was added with recombinant MBP-tagged PRMT1 protein in the presence or absence of S-adenosylmethionine (SAM). The methylation levels of FUS/TLS were monitored using anti-Dimethyl Arginine (DMA) antibody (upper panel). The membrane was reprobed with anti-GST antibody to check the loading volume of GST protein in each lane (lower panel). (B) In vivo methylation assay. Tet-on inducible PRMT1 over-expression HEK293 cells, treated with 1 µg/ml tetracycline (Tet+) or mock (Tet−) for 24 h, were lysed and immunoprecipitated with anti-FUS antibody. Then immunoprecipitates were blotted with anti-DMA antibody (upper panel). The membrane was reprobed with anti-FUS/TLS antibody to check the loading volume of FUS/TLS protein in each (middle panel). IgG, Immunoglobulin heavy chain. Western blots were scanned using Canon 8800F photo scanner, and densitometric analysis was performed using NIH Ima Graphs represent means of relative intensities in four experiments, corrected to total FUS bands detected by anti-FUS antibody, and expressed as fold over control (value 1). Error bars represent standard deviation. Student’s t -test was preformed to evaluate differences between two groups. * p <0.05 vs Tet-. (C) in vivo methylation assay. HEK293 cells were treated with PRMT1-targeted siRNA oligonucleotides (PRMT1 siRNA) or control siRNA (Cont siRNA) in a concentration of 40 nM for 24 h. Then lysates of HEK293 cells were immunoprecipitated with anti-FUS/TLS antibody, and the methylation levels of FUS/TLS were monitored with anti-DMA antibody (upper panel). Then membrane was reprobed with anti-FUS antibody to check the loading volume FUS/TLS protein in each lane (middle panel). IgG, Immunoglobulin heavy chain. Western blots were scanned using Canon 8800F photo scanner, and densitometric analysis was performed using NIH ImageJ. Graphs represent means of relative intensities in four experiments, corrected to total FUS bands detected by anti-FUS antibody, and expressed as fold over control (value 1). Error bars represent standard deviation. Student’s t -test was preformed to evaluate differences between two groups. * p <0.05 vs Cont siRNA.

Article Snippet: For in vitro methylation assay, recombinant His-MBP-tagged PRMT1 protein was obtained from ATGen Co. Ltd (Gyeonggi-do, Korea).

Techniques: In Vitro, Methylation, Control, Recombinant, Membrane, In Vivo, Over Expression, Immunoprecipitation, Western Blot, Standard Deviation, Concentration Assay