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Sino Biological hrv 3c protease
Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with <t>HRV</t> 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.
Hrv 3c Protease, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 91 stars, based on 1 article reviews
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1) Product Images from "Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination"

Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122293

Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.
Figure Legend Snippet: Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.

Techniques Used: Purification, Recombinant, Expressing, Concentration Assay, Fluorescence, Molecular Weight, Staining, SDS-Gel, Flow Cytometry, Size-exclusion Chromatography

Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.
Figure Legend Snippet: Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.

Techniques Used: Functional Assay, Expressing, Construct, Confocal Microscopy, Fluorescence, Binding Assay, Transformation Assay, Plasmid Preparation, Copurification, Isolation

Related Articles

Flow Cytometry:

Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination
Article Snippet: N-dodecyl β-D maltoside (DDM), N-decyl β-D maltoside (DM) and Fos-Choline-12 (FC-12) were from Anatrace. .. HRV 3C protease was from Sino Biological.

Affinity Chromatography:

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: Cells were harvested and lysed using a microfluidizer (Microfluidics, Inc.). .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.). .. Finally, GLIC or chimera were subjected to gel filtration on a Superdex 200 10/300 column (GE Healthcare) .

Isolation:

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: Cells were harvested and lysed using a microfluidizer (Microfluidics, Inc.). .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.). .. Finally, GLIC or chimera were subjected to gel filtration on a Superdex 200 10/300 column (GE Healthcare) .

Centrifugation:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: The lysate was cleared by centrifugation (17,000g × 20 min at 4 °C) and the supernatant was passed through a 45-μm mesh filter (Millex-HA, Merck-Millipore) before loading onto a GSH-Sepharose column (1 × 20 cm, GE Healthcare). .. The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Construct:

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: In brief, constructs were overexpressed in E. coli C41 cells after induction with IPTG overnight at 18°C. .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.).

Purification:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Paragraph title: Expression and purification of mouse recombinant PDE4B ... The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: Cells were harvested and lysed using a microfluidizer (Microfluidics, Inc.). .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.). .. Finally, GLIC or chimera were subjected to gel filtration on a Superdex 200 10/300 column (GE Healthcare) .

Incubation:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Fractions containing GST-PDE4B protein bands were pooled and concentrated using a 100-kD ultrafiltration unit (Amicon) while changing the buffer to enzyme storage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol and 10% (v/v) glycerol). .. The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol. .. Recombinant PDE4B (1 μg) was phosphorylated with 0.4 U of purified activated recombinant bacterially expressed AMPK or purified PKA catalytic subunits in 20 μl of kinase assay buffer (see PKA assay) containing 200 μM AMP (for AMPK only) and 100 μM [γ-32 P] ATP (specific radioactivity 1,000 c.p.m. pmol−1 ).

Recombinant:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Paragraph title: Expression and purification of mouse recombinant PDE4B ... The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Expressing:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Paragraph title: Expression and purification of mouse recombinant PDE4B ... The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: Paragraph title: GLIC wild-type and GLIC-5HT3A -ICD chimera expression in E. coli and purification ... Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.).

Protease Inhibitor:

Article Title: Production of chemokine/chemokine receptor complexes for structural and biophysical studies
Article Snippet: Centrifuge Kimble-Chase Kontes™ Dounce Tissue Grinders 100, 15, 1 mL (Fisher Scientific, 8853000100, 8853000040, 8853000001) Poly-Prep Chromatography Columns (Bio-rad, 7311550) Micro Bio-Spin Columns (Bio-rad, 7326204) PD-10 desalting column (GE Healthcare, 17-0851-01) .. cOmplete EDTA-free protease inhibitor tablets (Roche, 5056489001) Iodoacetamide (GE Healthcare, RPN6302) n-Dodecyl-β-D-Maltopyranoside (DDM) (Anatrace, D310) Cholesteryl hemisuccinate (CHS) (Sigma-Aldrich, C6512) TALON Superflow Metal Affinity Resin (Clontech, 635507) Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane (EMD Millipore, UFC510096) Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-100 membrane (EMD Millipore, UFC810096) HRV 3C Protease (Human Rhinovirus 3C Protease, PreScission Site) (Sino Biological, S3CP01) PNGaseF (New England Biolabs, P0704S) .. 10 mM HEPES pH 7.5 10 mM MgCl2 20 mM KCl cOmplete EDTA-free protease inhibitor tablets (Roche, 5056489001)

Lysis:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Bacteria were then collected by centrifugation (5,000g × 10 min at 4 °C), resuspended in 1/10 of the culture volume of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% (v/v) 2-mercaptoethanol, 0.01% (w/v) Brij 35, 0.5 mM phenylmethyl sulfonyl fluoride, 0.5 mM benzamidine Cl, 1 μg ml−1 leupeptin and 1 μg ml−1 aprotinin) and homogenized using a French press device. .. The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Crystallization Assay:

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: GLIC and GLIC-5-HT3A -ICD chimera were expressed and purified essentially as described previously for GLIC crystallization , . .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.).

Binding Assay:

Article Title: Direct interaction of the resistance to inhibitors of cholinesterase (RIC-3) protein with the serotonin receptor type 3A (5-HT3A) intracellular domain
Article Snippet: Cells were harvested and lysed using a microfluidizer (Microfluidics, Inc.). .. Membranes were isolated by ultracentrifugation, solubilized in a 2% n-dodecyl-β-D-maltoside (DDM) (Anatrace) buffer, purified by affinity chromatography (nickel or amylose) (New England Biolabs), and the maltose binding protein tag was cleaved with HRV-3C protease (Sino Biological Inc.). .. Finally, GLIC or chimera were subjected to gel filtration on a Superdex 200 10/300 column (GE Healthcare) .

Chromatin Immunoprecipitation:

Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination
Article Snippet: HRV 3C protease was from Sino Biological. .. HRV 3C protease was from Sino Biological.

SDS Page:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Fractions were subjected to SDS–PAGE followed by Coomassie Blue staining. .. The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

Plasmid Preparation:

Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination
Article Snippet: The pEGFP-N1 plasmid containing eGFP was purchased from Clontech. .. HRV 3C protease was from Sino Biological.

Staining:

Article Title: AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B
Article Snippet: Fractions were subjected to SDS–PAGE followed by Coomassie Blue staining. .. The GST tag was removed overnight at 4°C by incubation of 500 μg of fusion protein with 10U of HRV 3C protease (Sino Biological) according to the manufacturer's protocol.

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    Sino Biological recombinant hrv 3c protease
    Interruption of RIPK1 death signaling in <t>HRV-A16</t> infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and <t>3Cpro</t> from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.
    Recombinant Hrv 3c Protease, supplied by Sino Biological, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant hrv 3c protease/product/Sino Biological
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    Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Western Blot, Transfection, Expressing, Activation Assay, Binding Assay

    3Cpro generates a 60 kDa N-terminal RIPK1 fragment, and supports HRV-A16 titer production late in infection. a Western blots against RIPK1 (leucine 190), VP2, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) and treated with pan-caspase inhibitor QVD (5 µ m , 15 h), calpain/cathepsin inhibitor E64d (10 µ m , 15 h), necrostatin-1 (Nec1, 1 µ m , 15 h), AG7088 (rupintrivir, 20 n m , 15 h) or drug-combinations. The 60 kDa RIPK1 fragment is highlighted by black arrow. b In vitro cleavage of RIPK1 by 3Cpro. HeLa cell extracts were incubated with recombinant 3C protease (r3C) with or without the 3C inhibitor AG7088 (20 n m ), and analyzed by western blotting against RIPK1 (leucine 190) and lamin A/C. Reference samples included lysates from HeLa infected with HRV-A16 (MOI 1, 15 h). RIPK1 cleavage is highlighted by black arrows. c GST-RIPK1 cleavage in vitro. Recombinant GST-RIPK1 was incubated with r3C in presence or absence of AG7088 (20 n m ) or QVD (5 µ m ), and reaction products analyzed by western blotting against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 133–422), and GST. d Late addition of AG7088 inhibits production of HRV-A16. HeLa cells were infected with HRV-A16 at three different MOI (log −1, −2, −3) and treated with AG7088 at different times pi. The titer of viral progeny was determined from cells and supernatants 24 h pi for each time point by end point titration assays. Data points represent mean values from three different experiments, including SD.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: 3Cpro generates a 60 kDa N-terminal RIPK1 fragment, and supports HRV-A16 titer production late in infection. a Western blots against RIPK1 (leucine 190), VP2, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) and treated with pan-caspase inhibitor QVD (5 µ m , 15 h), calpain/cathepsin inhibitor E64d (10 µ m , 15 h), necrostatin-1 (Nec1, 1 µ m , 15 h), AG7088 (rupintrivir, 20 n m , 15 h) or drug-combinations. The 60 kDa RIPK1 fragment is highlighted by black arrow. b In vitro cleavage of RIPK1 by 3Cpro. HeLa cell extracts were incubated with recombinant 3C protease (r3C) with or without the 3C inhibitor AG7088 (20 n m ), and analyzed by western blotting against RIPK1 (leucine 190) and lamin A/C. Reference samples included lysates from HeLa infected with HRV-A16 (MOI 1, 15 h). RIPK1 cleavage is highlighted by black arrows. c GST-RIPK1 cleavage in vitro. Recombinant GST-RIPK1 was incubated with r3C in presence or absence of AG7088 (20 n m ) or QVD (5 µ m ), and reaction products analyzed by western blotting against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 133–422), and GST. d Late addition of AG7088 inhibits production of HRV-A16. HeLa cells were infected with HRV-A16 at three different MOI (log −1, −2, −3) and treated with AG7088 at different times pi. The titer of viral progeny was determined from cells and supernatants 24 h pi for each time point by end point titration assays. Data points represent mean values from three different experiments, including SD.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Western Blot, In Vitro, Incubation, Recombinant, Titration

    HRV-A16 infection disrupts an RHIM domain-containing RIPK1-cCasp8-p62/SQSTM1 signaling complex, and leads to complex formation of 60 kDa RIPK1-Caspase-8 and 3ABC/3Cpro. a Comparison of the RHIM domain from human RIPK1, RIPK3, 3Cpro from HRV-A16/B14 by sequence alignment. Yellow indicates amino acid identities, gray similarities, * denotes conserved amino acids in the RHIM domains of RIPK1/3. b Pro-caspase-8 immuno-precipitations, and western blots against Pro-caspase-8, RIPK1 (amino acids 190) and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), or poly I:C (5 µg/ml)-treated cells, and uninfected samples. Input lysates and immuno-precipitations are shown. LC denotes light chain. c RIPK1 immuno-precipitations (leucine 190) and western blot against 3Cpro from HRV-A16-infected HeLa cells (MOI 1, 15 h) or uninfected cells. HC denotes heavy chain. d RIPK1 immuno-precipitations (leucine 190) and western blots against RIPK1, cCasp8, and p62/SQSTM1 from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) or transfected with poly I:C (5 µg/ml). Input lysates and immuno-precipitations are shown. HC denotes heavy chain, IP immuno-precipitation. Arrow highlights HRV-A16-specific p62/SQSTM1 processing.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: HRV-A16 infection disrupts an RHIM domain-containing RIPK1-cCasp8-p62/SQSTM1 signaling complex, and leads to complex formation of 60 kDa RIPK1-Caspase-8 and 3ABC/3Cpro. a Comparison of the RHIM domain from human RIPK1, RIPK3, 3Cpro from HRV-A16/B14 by sequence alignment. Yellow indicates amino acid identities, gray similarities, * denotes conserved amino acids in the RHIM domains of RIPK1/3. b Pro-caspase-8 immuno-precipitations, and western blots against Pro-caspase-8, RIPK1 (amino acids 190) and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), or poly I:C (5 µg/ml)-treated cells, and uninfected samples. Input lysates and immuno-precipitations are shown. LC denotes light chain. c RIPK1 immuno-precipitations (leucine 190) and western blot against 3Cpro from HRV-A16-infected HeLa cells (MOI 1, 15 h) or uninfected cells. HC denotes heavy chain. d RIPK1 immuno-precipitations (leucine 190) and western blots against RIPK1, cCasp8, and p62/SQSTM1 from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) or transfected with poly I:C (5 µg/ml). Input lysates and immuno-precipitations are shown. HC denotes heavy chain, IP immuno-precipitation. Arrow highlights HRV-A16-specific p62/SQSTM1 processing.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Sequencing, Western Blot, Liquid Chromatography, Transfection, Immunoprecipitation

    Necrotic cell death rather than apoptosis in HRV-A16-infected primary human nasal epithelial cells. a Dose-dependent infection of hAECN with various A- and B-type rhinoviruses (HRV-A16, HRV-B14, HRV-B37, HRV-A2, HRV-A1A), and CVB3 and correlation with cell number. Values are means ± SD, n = 3. b Electron micrographs of hAECN infected with HRV-A16 (MOI 1) or treated with poly I:C or puromycin for 15 h, or uninfected cells. Proximity of vesicular structures with the cell surface indicated by black arrowheads, and swollen mitochondrial cristae in infected cells highlighted by arrows. c ROS production of HRV-A16 or HRV-A1A (MOI 1) infected cells 24 h pi, compared to luprox- (100 μ m , 1 h) or menadione (100 μ m , 1 h)-treated cells. d FACS analysis of annexin V stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. e Western blots against lamin A/C and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). M r denotes relative molecular weight in kDa. f FACS analysis propidium iodide (PI) stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. g Western blot analysis of caspase 3, 7, 8, 9 activations in hAECN after infection with HRV-A16 (MOI 1, 15 h), poly I:C transfection (5 μg/ml) or external poly I:C addition (ext, 50 μg/ml). Arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. Infection is indicated by blotting against 3Cpro using beta-tubulin as a loading control.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: Necrotic cell death rather than apoptosis in HRV-A16-infected primary human nasal epithelial cells. a Dose-dependent infection of hAECN with various A- and B-type rhinoviruses (HRV-A16, HRV-B14, HRV-B37, HRV-A2, HRV-A1A), and CVB3 and correlation with cell number. Values are means ± SD, n = 3. b Electron micrographs of hAECN infected with HRV-A16 (MOI 1) or treated with poly I:C or puromycin for 15 h, or uninfected cells. Proximity of vesicular structures with the cell surface indicated by black arrowheads, and swollen mitochondrial cristae in infected cells highlighted by arrows. c ROS production of HRV-A16 or HRV-A1A (MOI 1) infected cells 24 h pi, compared to luprox- (100 μ m , 1 h) or menadione (100 μ m , 1 h)-treated cells. d FACS analysis of annexin V stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. e Western blots against lamin A/C and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). M r denotes relative molecular weight in kDa. f FACS analysis propidium iodide (PI) stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. g Western blot analysis of caspase 3, 7, 8, 9 activations in hAECN after infection with HRV-A16 (MOI 1, 15 h), poly I:C transfection (5 μg/ml) or external poly I:C addition (ext, 50 μg/ml). Arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. Infection is indicated by blotting against 3Cpro using beta-tubulin as a loading control.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, FACS, Western Blot, Molecular Weight, Transfection

    TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Activation Assay, Infection, Western Blot, Transfection, Expressing, Incubation, Staining, Fluorescence, Microscopy

    Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.

    Journal: PLoS ONE

    Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

    doi: 10.1371/journal.pone.0122293

    Figure Lengend Snippet: Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.

    Article Snippet: HRV 3C protease was from Sino Biological.

    Techniques: Purification, Recombinant, Expressing, Concentration Assay, Fluorescence, Molecular Weight, Staining, SDS-Gel, Flow Cytometry, Size-exclusion Chromatography

    Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.

    Journal: PLoS ONE

    Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

    doi: 10.1371/journal.pone.0122293

    Figure Lengend Snippet: Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.

    Article Snippet: HRV 3C protease was from Sino Biological.

    Techniques: Functional Assay, Expressing, Construct, Confocal Microscopy, Fluorescence, Binding Assay, Transformation Assay, Plasmid Preparation, Copurification, Isolation

    SDS-PAGE analysis of the CD147 antigen prior to rabbit immunization. Eukaryotic-expressed recombinant protein CD147-Fc was treated with HRV 3C protease for 16 h, and the mixture was purified by Ni 2+ affinity chromatography followed by Protein A chromatography. SDS-PAGE analysis demonstrated that CD147 was of high purity and indicated that there was no residual Fc fragment or 3C protease. Lane 1, recombinant eukaryotic-expressed CD147-Fc, which has a dimer formation and has been partially degraded; Lane 2, the recombinant CD147-Fc protein was treated with HRV 3C protease for 16 h, resulting in a mixture of CD147-Fc, CD147, Fc fragments and HRV 3C protease; Lane 3, mixture purified by Ni 2+ affinity chromatography, which is not very powerful, and a certain amount of recombinant protein and HRV 3C protease remain; Lane 4, CD147 immunogen purified by Protein A chromatography, no recombinant protein or HRV 3C protease was detected; Lane M, protein molecular weight marker. HRV, human rhinovirus.

    Journal: Molecular Medicine Reports

    Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

    doi: 10.3892/mmr.2017.6523

    Figure Lengend Snippet: SDS-PAGE analysis of the CD147 antigen prior to rabbit immunization. Eukaryotic-expressed recombinant protein CD147-Fc was treated with HRV 3C protease for 16 h, and the mixture was purified by Ni 2+ affinity chromatography followed by Protein A chromatography. SDS-PAGE analysis demonstrated that CD147 was of high purity and indicated that there was no residual Fc fragment or 3C protease. Lane 1, recombinant eukaryotic-expressed CD147-Fc, which has a dimer formation and has been partially degraded; Lane 2, the recombinant CD147-Fc protein was treated with HRV 3C protease for 16 h, resulting in a mixture of CD147-Fc, CD147, Fc fragments and HRV 3C protease; Lane 3, mixture purified by Ni 2+ affinity chromatography, which is not very powerful, and a certain amount of recombinant protein and HRV 3C protease remain; Lane 4, CD147 immunogen purified by Protein A chromatography, no recombinant protein or HRV 3C protease was detected; Lane M, protein molecular weight marker. HRV, human rhinovirus.

    Article Snippet: The Fc fragment was cleaved by Human Rhinovirus (HRV) 3C Protease (Sino Biological, Inc., Beijing, China).

    Techniques: SDS Page, Recombinant, Flow Cytometry, Purification, Affinity Chromatography, Chromatography, Molecular Weight, Marker