Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Optimization of pGC-A expression in Sf9 cells. Western blot (anti-pGC-A) of total cell protein over different virus multiplicities of infection (MOI) and transfection time. MOI of 0 indicates no virus transfection. pGC-A (red arrow) is approximately 120 kDa.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Expressing, Western Blot, Infection, Transfection

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Determination of expressed full-length pGC-A functionality via whole-cell activity assay. The competitive ELISA assay measured the cGMP yield level in Sf9 cells expressing full-length pGC-A versus control Sf9 cells (n = 2). Both cell types were incubated with different concentrations of MANP ligand (0 to 10 7 pmol). Incubation with 0 pmol MANP served as a negative control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Activity Assay, Competitive ELISA, Expressing, Incubation, Negative Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purification of full-length pGC-A via affinity and size exclusion columns. ( A ) Western blot (anti-pGC-A) of samples from cell lysis to the affinity column. pGC-A (red arrow) is ~ 120 kDa. ( B ) Coomassie blue stain from cell lysis to the affinity column. ( C ) Coomassie blue stain of the eluted fraction from Superose 6 that was used for protein crystallization. M: marker; P: membrane pellet from ultracentrifugation after solubilization with n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS); FT: flowthrough from the affinity column; 50, 100, 500: imidazole (mM) at elution from the affinity column.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, Western Blot, Lysis, Affinity Column, Staining, Crystallization Assay, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Presence of pGC-A in crystals was confirmed via western blot. The anti-pGC-A antibody was used in the western blot. The pGC-A monomer is 120 kDa. Lane 1: combined mix: combined crystallization drops collected in the PCR tube. Lane 2: supernatant: the supernatant collected from the centrifuged combined mix. Lane 3: washed pellet: the crystal pellet was washed with the precipitant solution and collected again via centrifugation. Lane 4: washed supernatant: the supernatant from washed crystal pellet. Lane 5: crystallization drop: one crystallization hanging drop directly mixed with SDS sample buffer. Lane 6: crystallization sample: purified pGC-A before crystallization, serving as a positive control.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Western Blot, Crystallization Assay, Centrifugation, Purification, Positive Control

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Indexed diffraction patterns from pGC-A microcrystals collected via serial crystallography at the Advanced Photon Source. Three indexable diffraction patterns with the highest resolution of 3 Å. ( A ) Blue/pink diffraction graphs show diffraction patterns analyzed using CrystFEL software. ( B ) The original diffraction patterns shown with white background. All diffraction dots were manually circled in red for better visualization.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Software

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Dynamic oligomeric states of pGC-A seen in replicate runs of Superose 6 size exclusion chromatography may be dependent on protein concentration. ( A ) The Superose 6 10/300GL column performance profile. Five standard proteins were used to generate relative molecule elution points based on different molecular sizes. Thyroglobulin (669 kDa) eluted at 14.19 mL, ferritin (440 kDa) eluted at 15.96 mL, aldolase (158 kDa) eluted at 17.58 mL, ovalbumin (44 kDa) eluted at 18.49 mL, and aprotinin (6.5 kDa) eluted at 21.60 mL. ( B-D ) Size exclusion chromatography of pGC-A. ( B ) The peak intensity at 17.3 mL corresponds to the pGC-A monomeric state (120 kDa). pGC-A monomer is the major peak determined by chromatography. Other ratios were faded out in the background and served as supplemental comparison. ( C ) pGC-A tetramer and monomer present similar ratios in the chromatographic separation. The peak intensity at 14.76 mL and 17.29 mL corresponds to pGC-A tetrameric (480 kDa) and monomeric states, respectively. ( D ) The pGC-A tetramer is the major peak. The peak intensity at 15.05 mL corresponds to the pGC-A tetrameric state.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Size-exclusion Chromatography, Protein Concentration, Chromatography

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stain of high resolution clear-native PAGE from pGC-A Superose 6 fractions. Superose 6 column eluted tetramer (480 kDa) and monomer (120 kDa) size peaks were concentrated and analyzed in a 4–16% native gel. M, marker.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Silver Staining, Clear Native PAGE, Marker

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Silver stained clear-native PAGE of different oligomeric samples treated with or without dithiothreitol (DTT) overnight. Three peak samples, which represent the tetramer, dimer, and monomer of full-length pGC-A, were concentrated and split in half for treatment with or without DTT. S, concentrated sample only; S + DTT, concentrated sample incubated with 1 M DTT overnight.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Staining, Clear Native PAGE, Incubation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Purified full-length pGC-A in vitro functional activity test. ( A ) Functional activity for two pGC-A oligomeric states with ATP incubation. The control group was GTP and ATP in sample buffer. For activity values in units of mg of purified pGC-A, the y-axis values of pmol/mL can be converted to nmol/mg protein by multiplying by 0.00873. ( B ) Functional activity for two pGC-A oligomeric states without ATP incubation. The control group was GTP only in the sample buffer. ( C ) Competitive cGMP ELISA standard fit in four parameters logistic (4PL) curve. The left Y-axis is the B/B0 (%) value and represents the percentage of bound cGMP. The right Y-axis represents the average net optical density (OD) reading at 405 nm. Both standard curves were generated with a 95% confidence interval. ( D ) The cGMP yield differences between pGC-A oligomer samples incubated with or without ATP were analyzed. All raw data points were analyzed via the ROUT method (Q = 1%) to remove significantly impossible outlier values before data analysis. One-way ANOVA was used to determine the statistical significance between samples and control in graphs ( A ) and ( B ). Two-way ANOVA was used to determine the statistical significance in graph ( D ). Each dot represented to the sample point and plotted as mean ± standard deviation (SD). * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001 and ns P ≥ 0.05, Not significant.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Purification, In Vitro, Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation

Journal: Scientific Reports

Article Title: Purification, characterization, and preliminary serial crystallography diffraction advances structure determination of full-length human particulate guanylyl cyclase A receptor

doi: 10.1038/s41598-022-15798-z

Figure Lengend Snippet: Proposed native state and three-step mechanism of full-length pGC-A. First, the full-length pGC-A forms a tetramer complex in the native state by non-covalent interactions (e.g., hydrogen bond and hydrophobic interactions). In each tetramer complex, there are two functional units, and each functional unit may represent a dimer. The narrowest part of the tetramer is the transmembrane domain. Second, the pGC-A signal transduction mechanism is not ATP-dependent. The current ATP-dependent two-step activation mechanism should instead be three-step. The first step is ligand (ANP) binding, which moderately activates the pGC-A; the second step is binding ATP, which partially boosts protein activity; the third step is the pGC-A phosphorylation, which fully activates the guanylyl cyclase.

Article Snippet: The plasmid construct pFastBac1-pGC-A (Addgene #186626) was designed to allow expression in Sf9 insect cells of pGC-A (amino acids 33–1061 of NCBI Reference Sequence NP_000897.3).

Techniques: Functional Assay, Transduction, Activation Assay, Binding Assay, Activity Assay

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: The effect of time, temperature, and DNA/PEI ratio on mEGFP-MPP1 overexpression and HEK-293F cell viability and density. Flow cytometry analysis of the level of mEGFP-MPP1 overexpression after ( a ) 17 h transfection at 31 °C or ( b ) 5 h transfection at 37 °C using different DNA:PEI ratios. Error bars represent SD. Trypan blue-based analysis of the viability of cells transfected for ( c ) 17 h at 31 °C or ( d ) 5 h at 37 °C with different DNA:PEI ratios. The density of cells transfected for ( e ) 17 h at 31 °C or ( f ) 5 h at 37 °C with different DNA:PEI ratios. NT—non-transfected cells/ control; hpt—hours post-transfection. The procedure was performed in triplicate.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Over Expression, Flow Cytometry, Transfection, Control

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Expression and purification of MPP1 protein from HEK-293F cells. Western blot analysis of ( a ) crude cell lysates collected through transfection (0 h–72 h), samples collected after centrifugation (S1, P1) and purified recombinant protein (2 µg). The upper membrane was incubated with Anti-His-tag antibodies to detect overexpressed MPP1 and the lower membrane with Anti-GAPDH antibodies; ( b ) Coomassie blue staining of the SDS–PAGE gel with purified recombinant MPP1. The lower mass of purified recombinant MPP1 is a result of TAP-tag cleavage during the purification (for details, see ).

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Expressing, Purification, Western Blot, Transfection, Centrifugation, Recombinant, Membrane, Incubation, Staining, SDS Page

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Characterization of the purified recombinant MPP1 protein using circular dichroism spectroscopy. ( a ) CD spectra of MPP1 protein 1, 3, and 7 days after dialysis recorded at 20 °C. ( b ) Thermal denaturation of MPP1 protein reflected via changes in ellipticity at 222 nm. The MPP1 was stored at 4 °C. Fu—fraction unfolded.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Purification, Recombinant, Circular Dichroism, Spectroscopy

Journal: Membranes

Article Title: High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

doi: 10.3390/membranes11090715

Figure Lengend Snippet: Identification of palmitoylation of overexpressed recombinant MPP1 protein by the Acyl-RAC method. Identification of palmitoylation of ( a ) overexpressed MPP1 protein in HEK-293F lysates and ( b ) recombinant MPP1 purified from HEK-293F cells. Both membranes were incubated with Anti-His-tag antibodies to detect recombinant MPP1. IF—input fraction (sample taken before the cleavage step); pF—preserved fraction; cF—cleaved fraction; PR—preservative reagent; CR—cleavage reagent.

Article Snippet: An expression vector carrying a sequence encoding fused protein, mEGFP-MPP1 , was prepared by cloning the MPP1 sequence (NCBI Reference Sequence: NM_002436.3) into the mEGFP-HRas plasmid (Addgene Plasmid #18662) using XhoI and NotI restriction sites ( ).

Techniques: Recombinant, Purification, Incubation

Journal: Interdisciplinary Perspectives on Infectious Diseases

Article Title: The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases

doi: 10.1155/2022/1639990

Figure Lengend Snippet: Primers used for PCR analysis.

Article Snippet: The full length of a synthetic gene encoding hPVB19 NS1 protein (NCBI Reference Sequence: NC_000883.2) was received in the pCMV6-AC-GFP-NS1 constitutive expression vector (Biomatik, Ontario, Canada).

Techniques: Sequencing

Journal: Interdisciplinary Perspectives on Infectious Diseases

Article Title: The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases

doi: 10.1155/2022/1639990

Figure Lengend Snippet: (a) Expression of GFP in NS1-transfected HEK-293T cells was examined by fluorescence microscopy at (I) 24 hr (II) 48 hr (III) 72 hr posttransfection with 10x magnification and bar size of 25 μ m. (b) The expression of NS1 mRNA in HEK-293 T cells was examined and visualized on 1.5% agarose gel at 100 V for 40 min and stained with GelRed at the indicated times posttransfection. (c) Posttransfection expression levels of NS1 mRNA overtime. Green: after 24 hr, red: after 48 hr, blue: after 72 hr, brown: mock-transfection at 24, 48, and 72 hr posttransfection.

Article Snippet: The full length of a synthetic gene encoding hPVB19 NS1 protein (NCBI Reference Sequence: NC_000883.2) was received in the pCMV6-AC-GFP-NS1 constitutive expression vector (Biomatik, Ontario, Canada).

Techniques: Expressing, Transfection, Fluorescence, Microscopy, Agarose Gel Electrophoresis, Staining

Journal: Interdisciplinary Perspectives on Infectious Diseases

Article Title: The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases

doi: 10.1155/2022/1639990

Figure Lengend Snippet: Percentage of apoptotic cells in mock- and NS1-transfected HEK-293T cells at 24 hr, 48 hr, and 72 hr posttransfection was determined by flow cytometry of cells that stained double-positive for PE Annexin V and 7-AAD.

Article Snippet: The full length of a synthetic gene encoding hPVB19 NS1 protein (NCBI Reference Sequence: NC_000883.2) was received in the pCMV6-AC-GFP-NS1 constitutive expression vector (Biomatik, Ontario, Canada).

Techniques: Transfection, Flow Cytometry, Staining

Journal: Interdisciplinary Perspectives on Infectious Diseases

Article Title: The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases

doi: 10.1155/2022/1639990

Figure Lengend Snippet: Thirteen cytokines' concentration of mock-transfected, B19 NS1-transfected, and untransfected HEK-293T cell are measured at 24, 48, and 72 hr posttransfection. Cytokine concentrations are reported based on a picogram per milliliter as mean. The experiments were carried out two times in duplicate.

Article Snippet: The full length of a synthetic gene encoding hPVB19 NS1 protein (NCBI Reference Sequence: NC_000883.2) was received in the pCMV6-AC-GFP-NS1 constitutive expression vector (Biomatik, Ontario, Canada).

Techniques: Concentration Assay, Transfection