FluoroCalins: engineered lipocalins with novel binding functions fused to a fluorescent protein for applications in biomolecular imaging and detection

2019 ◽  
Vol 32 (6) ◽  
pp. 289-296 ◽  
Author(s):  
Evelyn Eggenstein ◽  
Antonia Richter ◽  
Arne Skerra
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Growth Factor Receptor ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Promising Alternative ◽  
Biomolecular Imaging

Abstract FluoroCalins represent novel bifunctional protein reagents derived from engineered lipocalins fused to a fluorescent reporter protein, here the enhanced green fluorescent protein (eGFP). We demonstrate the construction, facile bacterial production and broad applicability of FluoroCalins using two Anticalin® molecules directed against the tumor vasculature-associated extra domain B of fibronectin (ED-B) and the vascular endothelial growth factor receptor 3, a marker of tumor and lymphangiogenesis. FluoroCalins were prepared with two different spacers: (i) a short Ser3Ala linker and (ii) a long hydrophilic and conformationally unstructured PASylation® polypeptide comprising 200 Pro, Ala and Ser residues. These FluoroCalins were applied for direct target quantification in enzyme-linked immunosorbent assay as well as target detection by flow cytometry and fluorescence microscopy of live and fixed cells, respectively, demonstrating high specificity and signal-to-noise ratio. Hence, FluoroCalins offer a promising alternative to antibody-based reagents for state of the art fluorescent in vitro detection and biomolecular imaging.

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

scholarly journals ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Hang Yang ◽  
Yujing Gong ◽  
Huaidong Zhang ◽  
Irina Etobayeva ◽  
Paulina Miernikiewicz ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
High Specificity ◽  
Multidrug Resistant ◽  
Penicillin G ◽  
Dependent Manner ◽  
Pneumococcal Infections ◽  
Promising Alternative ◽  
Content Type ◽  
Conventional Antibiotics

ABSTRACT Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae. By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro. In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

scholarly journals Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins

2017 ◽  
Vol 2017 ◽  
pp. 1-14
Author(s):  
Mayra Rodríguez-Rodríguez ◽  
Rafael Herrera-Esparza ◽  
Juan-José Bollain y Goytia ◽  
María-Elena Pérez-Pérez ◽  
Deyanira Pacheco-Tovar ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Posttranslational Modifications ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Peptidylarginine Deiminase ◽  
Reporter Protein ◽  
Ductal Cells ◽  
Arginine Residues

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p<0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren’s syndrome.

scholarly journals Analysis of Proteasome-Dependent Proteolysis in Haloferax volcanii Cells, Using Short-Lived Green Fluorescent Proteins

2004 ◽  
Vol 70 (12) ◽  
pp. 7530-7538 ◽  
Author(s):  
Christopher J. Reuter ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Specific Inhibitor ◽  
Haloferax Volcanii ◽  
Reporter System ◽  
Reporter Protein ◽  
Protein Variant ◽  
Fluorescent Reporter ◽  
New Genes ◽  
Green Fluorescent

ABSTRACT Proteasomes are energy-dependent proteases that are central to the quality control and regulated turnover of proteins in eukaryotic cells. Dissection of this proteolytic pathway in archaea, however, has been hampered by the lack of substrates that are easily detected in whole cells. In the present study, we developed a convenient reporter system by functional expression of a green fluorescent protein variant with C-terminal fusions in the haloarchaeon Haloferax volcanii. The levels of this reporter protein correlated with whole-cell fluorescence that was readily detected in culture. Accumulation of the reporter protein was dependent on the sequence of the C-terminal amino acid fusion, as well as the presence of an irreversible, proteasome-specific inhibitor (clasto-lactacystin β-lactone). This inhibitor was highly specific for H. volcanii 20S proteasomes, with a Ki of ∼40 nM. In contrast, phenylmethanesulfonyl fluoride did not influence the levels of fluorescent reporter protein or inhibit 20S proteasomes. Together, these findings provide a powerful tool for the elucidation of protein substrate recognition motifs and the identification of new genes which may be involved in the proteasome pathway of archaea.

scholarly journals Novel Roles of Chloroquine and Hydroxychloroquine in Graves’ Orbitopathy Therapy by Targeting Orbital Fibroblasts

2020 ◽  
Vol 105 (6) ◽  
pp. 1906-1917 ◽  
Author(s):  
Yan Guo ◽  
Hai Li ◽  
Xueying Chen ◽  
Huasheng Yang ◽  
Hongyu Guan ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Fluorescent Protein ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

Abstract Context Graves’ orbitopathy (GO) causes infiltrative exophthalmos by inducing excessive proliferation, adipogenesis, and glycosaminoglycan production in orbital fibroblasts (OFs). Interference with OF autophagy is a potential therapy for proptosis. Objectives Here, we aimed to evaluate the effects of chloroquine (CQ) and hydroxychloroquine (HCQ), the autophagy inhibitors commonly used in clinical practice, on OFs. Design/Setting/Participants OFs isolated from patients with GO (GO-OFs) or control individuals (non-GO-OFs) were cultured in proliferation medium (PM) or subjected to differentiation medium. OFs were treated with CQ or HCQ (0, 0.5, 2, and 10 μM), and subsequently examined in vitro. Main Outcome Measures CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular viability. Adipogenesis was assessed with Western blot analysis, real-time polymerase chain reaction (PCR) , and Oil Red O staining. Hyaluronan production was determined by real-time PCR and enzyme-linked immunosorbent assay. Autophagy flux was detected through red fluorescent protein (RFP)-green fluorescent protein (GFP)-LC3 fluorescence staining and Western blot analyses. Results CQ/HCQ halted proliferation and adipogenesis in GO-OFs in a concentration-dependent manner through blockage of autophagy, phenotypes that were not detected in non-GO-OFs. The inhibitory effect of CQ/HCQ on hyaluronan secretion of GO-OFs was also concentration dependent, mediated by downregulation of hyaluronan synthase 2 rather than hyaluronidases. Moreover, CQ (10 μM) induced GO-OF apoptosis without aggravating oxidative stress. Conclusions The antimalarials CQ/HCQ affect proliferation, adipogenesis, and hyaluronan generation in GO-OFs by inhibiting autophagy, providing evidence that they can be used to treat GO as autophagy inhibitors.

Non-invasive bioluminescence imaging of caspase-3 activity in Breast Cancer

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14557-e14557
Author(s):  
C. C. Olsen ◽  
F. Li ◽  
Z. He ◽  
W. Li ◽  
C. Li
Keyword(s):  
Breast Cancer ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Fold Increase ◽  
Reporter System ◽  
Reporter Protein ◽  
Reporter Proteins

e14557 Background: Apoptosis is a major form of tumor cells death during cytotoxic therapy. Understanding the kinetics of apoptosis would greatly facilitate development of more effective therapeutic approaches. In order to monitor apoptosis activities in vivo, we developed a novel bioluminescence-based reporter gene to detect caspase 3 activities, which are elevated at the execution phase of apoptosis. Methods: A caspase-3 reporter system was constructed by combining two different reporter proteins; green fluorescent protein (GFP) and firefly luciferase (FL) linked through multiple polyubiquitin domains with a caspase-3 recognition site. Under normal circumstances, the reporter proteins are rapidly degraded by the proteasome system.. During apoptosis, activated caspse 3 cleaves off the multi-ubiquitin domain from the reporter protein. This enable the GFP and luciferase fusion reporter to be stabilized and achieve a significant gain in GFP protein and luciferase activities, which in turn could be monitored both in vitro and in vivo. 4T1 cells transduced with CMV-luc or Caspase-3 reporter xenografts were treated with both chemotherapy and radiation therapy and monitored for apoptosis activity. Results: In vitro experiments demonstrated increased luciferase with increasing radiation dose reflective of apoptosis with background levels nearly undetectable. Taxol was associated with a time-dependent increase from 24 to 72hrs after drug exposure, indicating that apoptosis is a gradual, heterogeneous process. EGFP signal increased from 1.85% in controls to 80.6% in cells treated with 1uM Taxol. Xenografts showed nearly undetectable luciferase background with Cytoxan therapy resulting in a 90-fold increase, 10 Gy a 24 fold increase and fractionated RT (5Gy x3) with a 46-fold increase. Conclusions: We developed a novel in vivo caspase reporter based on the ubiquitous proteosome system of protein degradation and bioluminsecence imaging. This allowed us to assess activation of apoptosis in response to chemoradiation therapy in tissue culture and breast cancer xenografts over the course of 2–3 weeks, which has not been possible with other technologies. No significant financial relationships to disclose.

scholarly journals Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

2007 ◽  
Vol 189 (18) ◽  
pp. 6564-6571 ◽  
Author(s):  
Jennifer S. Choy ◽  
Latt Latt Aung ◽  
A. Wali Karzai
Keyword(s):  
Messenger Rna ◽  
Fluorescent Protein ◽  
Protein Product ◽  
Lon Protease ◽  
Reporter Protein ◽  
Direct Role ◽  
Mutant Cells ◽  
Protein Tagging

ABSTRACT Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with λ-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

scholarly journals QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 165 ◽  
Author(s):  
Lynda Handala ◽  
Tony Fiore ◽  
Yves Rouillé ◽  
Francois Helle
Keyword(s):  
Viral Infections ◽  
Java Virtual Machine ◽  
High Content Screening ◽  
Supplementary Data ◽  
Reporter Protein ◽  
Fluorescent Reporter ◽  
Processing Program ◽  
Infected Cells ◽  
Number Of Cells

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

CLONING OF HUMAN INTERLEUKIN-10 GENE AND TRANSGENE EXPRESSION IN RABBIT SYNOVIAL CELLS IN VITRO

2008 ◽  
Vol 11 (01) ◽  
pp. 1-7 ◽  
Author(s):  
Xiangping Liu ◽  
Haibo Wang ◽  
Kun Yang ◽  
Aihua Sui ◽  
Yanming Wang ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Fluorescent Protein ◽  
Interleukin 10 ◽  
Eukaryotic Expression ◽  
Cell Culture Supernatant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Synovial Cells ◽  
Reading Frame ◽  
Green Fluorescent

In this work, the full-length open reading frame of the human interleukin-10 (hIL-10) gene was amplified through reverse transcription–polymerase chain reaction (RT-PCR), and then PCR products were inserted into pcDNA4/HisMax to construct an eukaryotic expression vector. After optimization by green fluorescent protein (GFP), recombinant hIL-10 genes were transfected and expressed in rabbit synovial cells compounded with liposome in vitro. In cell culture supernatant, rhIL-10 was detected using enzyme-linked immunosorbent assay (ELISA) at time intervals of 12 hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days. After 12 hours of transfection, ELISA showed that transgene expression of hIL-10 in rabbit synovial cells was elevated; at 72 hours, hIL-10 expression reached its peak value; and then it declined gradually until 7 days, compared with the control. After 14 days, transgene expression ceased. Gene cloning of hIL-10 and its transgene expression in synovial cells therefore gives a basis for the gene therapy of rheumatoid arthritis.

An anti-EGFR/anti- HER2 Bispecific Antibody with Enhanced Antitumor Activity Against Acquired Gefitinib-Resistant NSCLC Cells

2021 ◽  
Vol 28 ◽  
Author(s):  
Yan Si ◽  
Xinxin Pei ◽  
Xiangfang Wang ◽  
Qianqian Han ◽  
Changzhi Xu ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Kinase Inhibitors ◽  
Bispecific Antibody ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Akt Signaling ◽  
Enzyme Linked Immunosorbent Assay ◽  
Key Factor ◽  
Gefitinib Resistance

Background: Acquired resistance to epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKIs) is a recurrent phenomenon during clinical therapy of non‑small-cell lung cancer (NSCLC). Studies have shown that HER2 is a key factor contributing to drug resistance in a variety of cancers. Furthermore, we have observed that HER2 is overexpressed in PC-9 NSCLC cells with acquired gefitinib-resistance (PC-9/GR) as compared to that in PC-9 cells. Objective: We hypothesized that blocking both EGFR and HER2 may serve as a potential strategy for treatment of NSCLC with acquired gefitinib-resistance. Methods: To target both EGFR and HER2 simultaneously, we developed a bispecific antibody HECrossMAb, which was derived from a humanized Cetuximab and Trastuzumab. The binding affinity of HECrossMAb for EGFR and HER2 was measured using enzyme-linked immunosorbent assay. The MTT assay was used to determine the effect of HECrossMAb on the proliferation of PC‑9 and PC‑9/GR cells in vitro. Finally, the effect of HECrossMAb on PI3K/AKT signaling and associated transcription factors was measured using western blot analysis. Results: Our results showed that HECrossMAb exerts enhanced cytotoxicity in both PC-9 and PC-9/GR cells by inhibiting the activation of PI3K/AKT signaling and expression of relevant transcription factors such as AEG-1, c-Myc, and c-Fos. Conclusion: Our results suggest that HECrossMAb may function as a potential therapeutic agent for the treatment of NSCLC overexpressing EGFR and HER2.

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