Long-circulating XTEN864-annexin A5 fusion protein for phosphatidylserine-related therapeutic applications

AbstractAnnexin A5 (anxA5) is a marker for apoptosis, but has also therapeutic potential in cardiovascular diseases, cancer, and, due to apoptotic mimicry, against dangerous viruses, which is limited by the short blood circulation. An 864-amino-acid XTEN polypeptide was fused to anxA5. XTEN864-anxA5 was expressed in Escherichia coli and purified using XTEN as tag. XTEN864-anxA5 was coupled with DTPA and indium-111. After intravenous or subcutaneous injection of 111In-XTEN864-anxA5, mouse blood samples were collected for blood half-life determination and organ samples for biodistribution using a gamma counter. XTEN864-anxA5 was labeled with 6S-IDCC to confirm binding to apoptotic cells using flow cytometry. To demonstrate targeting of atherosclerotic plaques, XTEN864-anxA5 was labeled with MeCAT(Ho) and administered intravenously to atherosclerotic ApoE−/− mice. MeCAT(Ho)-XTEN864-anxA5 was detected together with MeCAT(Tm)-MAC-2 macrophage antibodies by imaging mass cytometry (CyTOF) of aortic root sections. The ability of anxA5 to bind apoptotic cells was not affected by XTEN864. The blood half-life of XTEN864-anxA5 was 13 h in mice after IV injection, markedly longer than the 7-min half-life of anxA5. 96 h after injection, highest amounts of XTEN864-anxA5 were found in liver, spleen, and kidney. XTEN864-anxA5 was found to target the adventitia adjacent to atherosclerotic plaques. XTEN864-anxA5 is a long-circulating fusion protein that can be efficiently produced in E. coli and potentially circulates in humans for several days, making it a promising therapeutic drug.

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Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2011.4.3.8 ◽

2011 ◽

Vol 4 (3) ◽

pp. 1491-1496

Author(s):

Umadevi M ◽

Rani T ◽

Balakrishnan T ◽

Ramanibai R

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Silver Nanoparticles ◽

Reduction Method ◽

Therapeutic Potential ◽

Chemical Reduction ◽

Ultrasonic Field ◽

Great Promise ◽

Chemical Reduction Method ◽

E Coli

Nanotechnology has great promise for improving the therapeutic potential of medicinal molecules and related agents. In this study, silver nanoparticles of different sizes were synthesized in an ultrasonic field using the chemical reduction method with sodium borohydride as a reducing agent. The size effect of silver nanoparticles on antimicrobial activity were tested against the microorganisms Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934). The results shows that B. subtilis, and E. coli were more sensitive to silver nanoparticles and its size, indicating the superior antimicrobial efficacy of silver nanoparticles.

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Expression of MBP-HCV NS1/E2 fusion protein in E. coli and detection of anti-NS1/E2 antibody in type c chronic liver disease

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(05)80278-5 ◽

1992 ◽

Vol 183 (3) ◽

pp. 925-930 ◽

Cited By ~ 14

Author(s):

Eiji Mita ◽

Norio Hayashi ◽

Keiji Ueda ◽

Akinori Kasahara ◽

Hideyuki Fusamoto ◽

...

Keyword(s):

Liver Disease ◽

Fusion Protein ◽

Chronic Liver Disease ◽

Chronic Liver ◽

E Coli ◽

Type C

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Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0612 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1461-1468 ◽

Cited By ~ 2

Author(s):

Jean-Claude Alvarez ◽

Pierre Moine ◽

Isabelle Etting ◽

Djillali Annane ◽

Islam Amine Larabi

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Half Life ◽

Limit Of Detection ◽

Treated Patient ◽

Internal Standard ◽

Therapeutic Drug ◽

Active Metabolites ◽

Limit Of Quantification ◽

Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

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Feasibility of Extrapolating Randomly Taken Plasma Samples to Trough Levels for Therapeutic Drug Monitoring Purposes of Small Molecule Kinase Inhibitors

Pharmaceuticals ◽

10.3390/ph14020119 ◽

2021 ◽

Vol 14 (2) ◽

pp. 119

Author(s):

Ruben A. G. van Eerden ◽

Esther Oomen-de Hoop ◽

Aad Noordam ◽

Ron H. J. Mathijssen ◽

Stijn L. W. Koolen

Keyword(s):

Therapeutic Drug Monitoring ◽

Small Molecule ◽

Trough Concentration ◽

Drug Monitoring ◽

Half Life ◽

Kinase Inhibitors ◽

Therapeutic Drug ◽

Blood Samples ◽

Elimination Half ◽

Trough Levels

Small molecule kinase inhibitors (SMKIs) are widely used in oncology. Therapeutic drug monitoring (TDM) for SMKIs could reduce underexposure or overexposure. However, logistical issues such as timing of blood withdrawals hamper its implementation into clinical practice. Extrapolating a random concentration to a trough concentration using the elimination half-life could be a simple and easy way to overcome this problem. In our study plasma concentrations observed during 24 h blood sampling were used for extrapolation to trough levels. The objective was to demonstrate that extrapolation of randomly taken blood samples will lead to equivalent estimated trough samples compared to measured Cmin values. In total 2241 blood samples were analyzed. The estimated Ctrough levels of afatinib and sunitinib fulfilled the equivalence criteria if the samples were drawn after Tmax. The calculated Ctrough levels of erlotinib, imatinib and sorafenib met the equivalence criteria if they were taken, respectively, 12 h, 3 h and 10 h after drug intake. For regorafenib extrapolation was not feasible. In conclusion, extrapolation of randomly taken drug concentrations to a trough concentration using the mean elimination half-life is feasible for multiple SMKIs. Therefore, this simple method could positively contribute to the implementation of TDM in oncology.

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Characterization of Anti-Bacterial Effect of the Two New Phages against Uropathogenic Escherichia coli

Viruses ◽

10.3390/v13071348 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1348

Author(s):

Lívia Slobodníková ◽

Barbora Markusková ◽

Michal Kajsík ◽

Michal Andrezál ◽

Marek Straka ◽

...

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Therapeutic Potential ◽

Medical Intervention ◽

Uropathogenic Escherichia Coli ◽

E Coli ◽

Phage Cocktail ◽

Causative Agents ◽

Tract Infections

Urinary tract infections (UTIs) are among the events that most frequently need medical intervention. Uropathogenic Escherichia coli are frequently their causative agents and the infections are sometimes complicated by the presence of polyresistant nosocomial strains. Phage therapy is a tool that has good prospects for the treatment of these infections. In the present study, we isolated and characterized two bacteriophages with broad host specificity against a panel of local uropathogenic E. coli strains and combined them into a phage cocktail. According to genome sequencing, these phages were closely related and belonged to the Tequatrovirus genus. The newly isolated phages showed very good activity on a panel of local clinical E. coli strains from urinary tract infections. In the form of a two-phage cocktail, they were active on E. coli strains belonging to phylogroups B2 and D, with relatively lower activity in B1 and no response in phylogroup A. Our study is a preliminary step toward the establishment of a national phage bank containing local, well-characterized phages with therapeutic potential for patients in Slovakia.

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Expression and partial purification of human pro-opiomelanocortin in Escherichia coli

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0030105 ◽

1989 ◽

Vol 3 (2) ◽

pp. 105-112 ◽

Cited By ~ 5

Author(s):

T. S. Grewal ◽

P. J. Lowry ◽

D. Savva

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Fusion Protein ◽

Western Blot ◽

Blot Analysis ◽

Expression Vectors ◽

Partial Purification ◽

Amino Acid Residues ◽

E Coli ◽

Dna Fragment

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

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Development of Stable Chimeric IL-15 for Trans-Presentation by the Antigen Presenting Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.646159 ◽

2021 ◽

Vol 12 ◽

Author(s):

Manoj Patidar ◽

Naveen Yadav ◽

Sarat K. Dalai

Keyword(s):

T Cells ◽

T Cell ◽

Half Life ◽

Therapeutic Potential ◽

Chimeric Protein ◽

Antigen Presenting Cells ◽

T Cell Response ◽

Antigen Presenting

IL-15 is one of the important biologics considered for vaccine adjuvant and treatment of cancer. However, a short half-life and poor bioavailability limit its therapeutic potential. Herein, we have structured IL-15 into a chimeric protein to improve its half-life enabling greater bioavailability for longer periods. We have covalently linked IL-15 with IgG2 base to make the IL-15 a stable chimeric protein, which also increased its serum half-life by 40 fold. The dimeric structure of this kind of IgG based biologics has greater stability, resistance to proteolytic cleavage, and less frequent dosing schedule with minimum dosage for achieving the desired response compared to that of their monomeric forms. The structured chimeric IL-15 naturally forms a dimer, and retains its affinity for binding to its receptor, IL-15Rβ. Moreover, with the focused action of the structured chimeric IL-15, antigen-presenting cells (APC) would transpresent chimeric IL-15 along with antigen to the T cell, that will help the generation of quantitatively and qualitatively better antigen-specific memory T cells. In vitro and in vivo studies demonstrate the biological activity of chimeric IL-15 with respect to its ability to induce IL-15 signaling and modulating CD8+ T cell response in favor of memory generation. Thus, a longer half-life, dimeric nature, and anticipated focused transpresentation by APCs to the T cells will make chimeric IL-15 a super-agonist for memory CD8+ T cell responses.

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Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.7412 ◽

2015 ◽

Vol 10 (1) ◽

Author(s):

Aris Haryanto

Keyword(s):

Fusion Protein ◽

Nuclear Import ◽

Recombinant Dna ◽

Core Protein ◽

Intracellular Localization ◽

Full Length ◽

Nuclear Pore ◽

E Coli ◽

Importin Α ◽

Localization Signal

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

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Improved CUT&RUN chromatin profiling and analysis tools

10.1101/569129 ◽

2019 ◽

Cited By ~ 1

Author(s):

Michael P. Meers ◽

Terri Bryson ◽

Steven Henikoff

Keyword(s):

Fusion Protein ◽

High Efficiency ◽

Low Cost ◽

Protein A ◽

Micrococcal Nuclease ◽

Permeabilized Cells ◽

E Coli ◽

Carry Over ◽

Chromatin Profiling ◽

Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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