Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture

Poly(A)+ RNA was isolated from human prostatic tissue and translated in vitro in a rabbit reticulocyte lysate translation assay. Acid phosphatase labeled with [35S]methionine was immunoprecipitated with an antibody against seminal plasma acid phosphatase. Two-dimensional polyacrylamide gel electrophoresis of the immunoprecipitate, followed by fluorography, revealed the presence of two spots (one major and one minor), both having a molecular mass of 43 kilodaltons (kDa) and an isoelectric point higher than mature acid phosphatase. Addition of canine pancreatic membranes to the translation assay resulted in the formation of four immunoprecipitable spots with molecular masses ranging from 43 to 49 kDa on one-dimensional gels. These spots probably represent acid phosphatases containing one to four core sugar groups, since after the addition of endoglycosidase H the molecular mass heterogeneity was abolished and we observed only one major band with a molecular mass (41 kDa) slightly lower than the ones of the primary translation product. These results suggest that human prostatic acid phosphatases are synthesized as two 43-kDa preproteins, which are further processed to 41-kDa proteins by removal of their signal peptide. Heterogeneity of the native protein arises mostly from glycosylation at four sites and not from differences in the amino acid sequence of the various forms.

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Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2

Frontiers in Plant Science ◽

10.3389/fpls.2020.604663 ◽

2021 ◽

Vol 11 ◽

Author(s):

Konlavat Siriwattananon ◽

Suwimon Manopwisedjaroen ◽

Phongthon Kanjanasirirat ◽

Priyo Budi Purwono ◽

Kaewta Rattanapisit ◽

...

Keyword(s):

Fusion Protein ◽

Vero Cells ◽

Global Public Health ◽

Potential Threat ◽

Angiotensin Converting Enzyme 2 ◽

Therapeutic Drugs ◽

Fc Fusion Protein ◽

Huge Impact ◽

Human Igg1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19) which has recently emerged as a potential threat to global public health. SARS-CoV-2 is the third known human coronavirus that has huge impact on the human population after SARS-CoV and MERS-CoV. Although some vaccines and therapeutic drugs are currently in clinical trials, none of them are approved for commercial use yet. As with SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the cell entry receptor to enter into the host cell. In this study, we have transiently produced human ACE2 fused with the Fc region of human IgG1 in Nicotiana benthamiana and the in vitro neutralization efficacy of the plant-produced ACE2-Fc fusion protein was assessed. The recombinant ACE2-Fc fusion protein was expressed in N. benthamiana at 100 μg/g leaf fresh weight on day 6 post-infiltration. The recombinant fusion protein showed potent binding to receptor binding domain (RBD) of SARS-CoV-2. Importantly, the plant-produced fusion protein exhibited potent anti-SARS-CoV-2 activity in vitro. Treatment with ACE2-Fc fusion protein after viral infection dramatically inhibit SARS-CoV-2 infectivity in Vero cells with an IC50 value of 0.84 μg/ml. Moreover, treatment with ACE2-Fc fusion protein at the pre-entry stage suppressed SARS-CoV-2 infection with an IC50 of 94.66 μg/ml. These findings put a spotlight on the plant-produced ACE2-Fc fusion protein as a potential therapeutic candidate against SARS-CoV-2.

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High-Throughput Screen Identifies Cyclic Nucleotide Analogs That Inhibit Prostatic Acid Phosphatase

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112468613 ◽

2012 ◽

Vol 18 (4) ◽

pp. 481-489 ◽

Cited By ~ 2

Author(s):

Eric S. McCoy ◽

Wendy A. Lea ◽

Bryan T. Mott ◽

David J. Maloney ◽

Ajit Jadhav ◽

...

Keyword(s):

Acid Phosphatase ◽

High Throughput ◽

Cyclic Nucleotide ◽

Prostatic Acid Phosphatase ◽

Live Cells ◽

Nucleotide Analogs ◽

Small Molecule Modulators ◽

Pharmacologically Active ◽

Kinetic Mode

The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well–based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.

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Recombinant canine IL-13 receptor α2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Veterinary Immunology and Immunopathology ◽

10.1016/s0165-2427(01)00378-6 ◽

2001 ◽

Vol 83 (1-2) ◽

pp. 115-122 ◽

Cited By ~ 5

Author(s):

Liang Tang ◽

Karen L. Boroughs ◽

Tony Morales ◽

Kim Stedman ◽

Karen Sellins ◽

...

Keyword(s):

Fusion Protein ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Specific Ige ◽

Peripheral Blood Mononuclear ◽

Fc Fusion Protein ◽

Blood Mononuclear Cells

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Prostatic Acid Phosphatase-Sargramostim Fusion Protein PA2024

10.32388/a8baew ◽

2020 ◽

Author(s):

Keyword(s):

Acid Phosphatase ◽

Fusion Protein ◽

Prostatic Acid Phosphatase

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The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia

Blood ◽

10.1182/blood-2009-05-217513 ◽

2009 ◽

Vol 114 (26) ◽

pp. 5362-5367 ◽

Cited By ~ 57

Author(s):

Xiao-juan Zhu ◽

Yan Shi ◽

Jun Peng ◽

Cheng-shan Guo ◽

Ning-ning Shan ◽

...

Keyword(s):

Fusion Protein ◽

Immune Thrombocytopenia ◽

Interleukin 4 ◽

In Vitro Assays ◽

Enzyme Linked Immunosorbent Assay ◽

Cd8 Cells ◽

Promising Therapeutic Approach ◽

Fc Fusion Protein ◽

Ifn Γ

Abstract Elevated level of B-cell activating factor (BAFF) has been implicated in the pathogenesis of some autoimmune diseases. Blockade of receptor and ligand binding by decoy receptor has demonstrated a clinical benefit in both oncologic and immunologic diseases. In this report, we have detected plasma BAFF and BAFF mRNA expression in immune thrombocytopenia (ITP) patients by enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR). The effects of recombinant human BAFF (rhBAFF) and BAFF-R-Fc fusion protein (BR3-Fc) on B cells, T cells, platelets, secretion of interferon γ (IFNγ), and interleukin-4 (IL-4) were measured by flow cytometry and ELISA. Patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. In in vitro assays, rhBAFF promoted the survival of CD19+ and CD8+ cells, and increased the apoptosis of platelets and the secretion of IFN-γ. BR3-Fc successfully corrected the effects of rhBAFF on lymphocytes, platelets, and cytokines. These findings suggest that BAFF may play a pathogenic role in ITP by promoting the survival of CD19+ and CD8+ cells, and increasing the apoptosis of platelets and the secretion of IFN-γ. Blockade of BAFF by BR3-Fc might be a promising therapeutic approach for ITP.

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