Challenges with development of a pharmacokinetics assay to measure a variably glycosylated fusion protein

Bioanalysis ◽  
2021 ◽  
Author(s):  
Gizette Sperinde ◽  
Luna Liu ◽  
Keyang Xu ◽  
Tracy Bentley ◽  
Sid Sukumaran ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Clinical Sample ◽  
High Sensitivity ◽  
Matrix Interference ◽  
Method Transfer ◽  
Recombinant Fusion Proteins ◽  
Drug Concentrations

Aim: Development of recombinant fusion proteins as drugs poses unique challenges for bioanalysis. This paper describes a case study of a glycosylated fusion protein, where variable glycosylation, matrix interference and high sensitivity needs posed unique challenges. Results: Six different assay configurations, across four different platforms were evaluated for measurement of drug concentrations. Two platforms that achieved the assay requirements were Simoa HD-1 and immune-capture LC–MS/MS-based assay. Conclusion: Both, Simoa HD-1 and the mass spectrometry-based methods were able to detect total drug by providing the adequate matrix tolerance, required sensitivity and detection of all the various glycosylated fusion proteins to support clinical sample analysis. The mass spectrometry-based method was selected due to robustness and ease of method transfer.

scholarly journals DEVELOPMENT OF THE VACCINE BASED ON THE RECOMBINANT ANTIGENS OF PSEUDOMONAS AERUGINOSA

2019 ◽  
Vol 1 (1) ◽  
pp. 74-80
Author(s):  
N. A. Mihailova ◽  
E. M. Zimina ◽  
A. V. Soldatenkova ◽  
A. A. Kaloshin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Fusion Protein ◽  
Outer Membrane ◽  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Outer Membrane Proteins ◽  
Recombinant Fusion Protein ◽  
Recombinant Antigens ◽  
Recombinant Fusion Proteins

Aim. The aim is obtaining, investigation and selection of recombinant antigens for inclusion theirs into the against Pseudomonas vaccine. Materials and methods. The genes encoding of the outer  membrane proteins F, L and I and Exotoxin A were synthesized by PCR with the genomic DNA of Pseudomonas aeruginosa. The amplified sequences were cloned into plasmid vectors for expression in cells of Escherichia coli. As the result of expression were the synthesized recombinant proteins that were purified in columns with a nickel-activated sorbent. The authenticity of the recombinant antigens was assessed by electrophoresis and immunoblotting. For assessing the immunogenicity of the recombinant proteins,they were sorbed on aluminum hydroxide and used for intraperitoneal immunization of mice. After a course of immunization, mice were injected intraperitoneally with a live virulent culture or еxotoxin A. Results. The obtained recombinant outer membrane proteins OprF, OprL and OprI, as well as the deletion variant of еxotoxin A (toxoid) stimulated immune reactions and protected the experimental animals from the virulent culture of P. aeruginosa. Using of the complexes of the recombinant proteins, as well as immunization with the fusion proteins consisting from sequences of two or three recombinant antigens, produced an additive increase in protective effects. The combination of the recombinant OprF protein and the recombinant toxoid (efficiency index of protective properties (EI 3.0) and two recombinant fusion proteins (EI 3.5) were the most effective. The first recombinant fusion protein (OprF-aTox-OprI) consisted from fused polypeptide sequences of OprF, toxoid and OprI. The second recombinant fusion protein (OprF-OprI) consisted from fused polypeptide sequences of OprF and OprI. Conclusion. The data obtained showed the fundamental possibility of using recombinant fusion proteins OprF-aTox-OprI and OprF-OprI as well as the complex of the recombinant OprF protein and the recombinant toxoid as the candidated vaccines against Pseudomonas aeruginosa.

scholarly journals Expression in Bacteria of the Gene Encoding the gp43 Antigen of Paracoccidioides brasiliensis: Immunological Reactivity of the Recombinant Fusion Proteins

2002 ◽  
Vol 9 (6) ◽  
pp. 1200-1204 ◽  
Author(s):  
Susana N. Diniz ◽  
Kátia C. Carvalho ◽  
Patrícia S. Cisalpino ◽  
José F. Silveira ◽  
Luiz R. Travassos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Paracoccidioides Brasiliensis ◽  
Immunological Reactivity ◽  
Gene Encoding ◽  
Recombinant Fusion Proteins ◽  
Gst Fusion Protein

ABSTRACT gp43 is the major diagnostic antigen of Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis (PCM) in humans. In the present study, cDNA of the gp43 gene (PbGP43) was obtained by reverse transcriptase PCR, inserted into a pGEX vector in frame with the glutathione S-transferase (GST) gene, and expressed in Escherichia coli as inclusion bodies. Immunoblotting showed that all sera from patients with chronic pulmonary and acute lymphatic forms of PCM reacted with the recombinant fusion protein of the mature gp43 (381 amino acids). Reactivity with fusion proteins containing subfragments of the N-terminal, internal, or C-terminal regions occurred eventually, and the C-terminal region was the most antigenic. Lack of reactivity with the subfragments may be due to the conformational nature of the gp43 epitopes. Sera from patients with aspergillosis, candidiasis, and histoplasmosis did not react with the gp43-GST fusion protein. Our results suggest that recombinant gp43 corresponding to the processed antigen can be a useful tool in the diagnosis of PCM.

scholarly journals Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

1999 ◽  
Vol 6 (2) ◽  
pp. 168-172 ◽  
Author(s):  
Y. Abed ◽  
G. St-Laurent ◽  
H. Zhang ◽  
R. M. Jacobs ◽  
D. Archambault
Keyword(s):  
Recombinant Protein ◽  
Fusion Protein ◽  
Western Blot ◽  
Fusion Proteins ◽  
Transmembrane Protein ◽  
Recombinant Baculovirus ◽  
Serum Samples ◽  
Western Blot Assay ◽  
Recombinant Fusion Proteins ◽  
Syncytial Virus

ABSTRACT A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.

scholarly journals Biomolecular Condensation Drives Leukemia Caused by NUP98-Fusion Proteins

2020 ◽  
Author(s):  
Stefan Terlecki-Zaniewicz ◽  
Thomas Eder ◽  
Johannes Schmöllerl ◽  
Theresa Humer ◽  
Natalie Kuchynka ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Affinity Purification ◽  
Structural Features ◽  
Gene Control ◽  
Intrinsically Disordered ◽  
Epigenetic Regulators

AbstractNUP98-fusion proteins cause acute myeloid leukemia via unknown molecular mechanisms. All NUP98-fusion proteins share an intrinsically disordered region (IDR) featuring >35 repeats of Phenylalanine-Glycine (FG) in the NUP98 N-terminus. Conversely, different C-terminal NUP98-fusion partners are often transcriptional and epigenetic regulators. Given these structural features we hypothesized that mechanisms of oncogenic transformation by NUP98-fusion proteins are hard-wired in their protein interactomes. Affinity purification coupled to mass spectrometry of five distinct NUP98-fusion proteins revealed a conserved set of interactors that was highly enriched for proteins involved in biomolecular condensation. We developed biotinylated isoxazole-mediated condensome mass spectrometry (biCon-MS) to show that NUP98-fusion proteins alter the global composition of biomolecular condensates. In addition, an artificial FG-repeat containing fusion protein was able to phenocopy the induction of leukemic gene expression as mediated by NUP98-KDM5A. Thus, we propose that IDR-containing fusion proteins have evolved to uniquely combine biomolecular condensation with gene control to induce cancer.AML, NUP98, fusion protein, AP-MS, LLPS, biCon-MS, condensate

Staphylokinase as a Plasminogen Activator Component in Recombinant Fusion Proteins

1999 ◽  
Vol 65 (2) ◽  
pp. 506-513 ◽  
Author(s):  
S. J. Szarka ◽  
E. G. Sihota ◽  
H. R. Habibi ◽  
S.-L. Wong
Keyword(s):  
Fusion Protein ◽  
Plasminogen Activator ◽  
Fusion Proteins ◽  
Site Directed Mutagenesis ◽  
Thrombin Inhibitor ◽  
Fusion Partner ◽  
Resistant Variant ◽  
Recombinant Fusion Proteins ◽  
Culture Supernatants

ABSTRACT The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

Algorithm of combining chromatography mass spectrometry-untargeted profiling and multivariate analysis for identification of marker-substances in samples of complex composition

2020 ◽  
Vol 86 (7) ◽  
pp. 12-19
Author(s):  
I. V. Plyushchenko ◽  
D. G. Shakhmatov ◽  
I. A. Rodin
Keyword(s):  
Mass Spectrometry ◽  
Multivariate Analysis ◽  
Large Scale ◽  
Complex Composition ◽  
Unified Protocol ◽  
Chromatography Mass Spectrometry ◽  
Marker Substances ◽  
Selection Testing ◽  
Untargeted Profiling

A viral development of statistical data processing, computing capabilities, chromatography-mass spectrometry, and omics technologies (technologies based on the achievements of genomics, transcriptomics, proteomics, metabolomics) in recent decades has not led to formation of a unified protocol for untargeted profiling. Systematic errors reduce the reproducibility and reliability of the obtained results, and at the same time hinder consolidation and analysis of data gained in large-scale multi-day experiments. We propose an algorithm for conducting omics profiling to identify potential markers in the samples of complex composition and present the case study of urine samples obtained from different clinical groups of patients. Profiling was carried out by the method of liquid chromatography mass spectrometry. The markers were selected using methods of multivariate analysis including machine learning and feature selection. Testing of the approach was performed using an independent dataset by clustering and projection on principal components.

scholarly journals A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 468
Author(s):  
Anthony E. Jones ◽  
Nataly J. Arias ◽  
Aracely Acevedo ◽  
Srinivasa T. Reddy ◽  
Ajit S. Divakaruni ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Biosynthetic Pathway ◽  
Solid Phase ◽  
High Sensitivity ◽  
Short Chain ◽  
Extraction Column ◽  
Chain Acyl ◽  
Precision And Accuracy ◽  
Mass Spectrometry Mass Spectrometry

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels.

scholarly journals Conducting Web-Based Focus Groups With Adolescents and Young Adults

2021 ◽  
Vol 20 ◽  
pp. 160940692199687
Author(s):  
Courtney A. Brown ◽  
Anna C. Revette ◽  
Sarah D. de Ferranti ◽  
Holly B. Fontenot ◽  
Holly C. Gooding
Keyword(s):  
Qualitative Research ◽  
Young Adults ◽  
Focus Groups ◽  
Clinical Sample ◽  
Adolescents And Young Adults ◽  
Web Based ◽  
Disease Awareness ◽  
Sex With Men ◽  
Technical Platform

This methodologic paper aims to update researchers working with adolescents and young adults on the potentials and pitfalls associated with web-based qualitative research. We present a case study of synchronous web-based focus groups with 35 adolescents and young women ages 15–24 years old recruited from a clinical sample for a mixed methods study of heart disease awareness. We contrast this with two other studies, one using asynchronous web-based focus groups with 30 transgender youth ages 13 to 24 years old and another using synchronous web-based focus groups with 48 young men who have sex with men ages 18 to 26 years old, both recruited via social media. We describe general and logistical considerations, technical platform considerations, and ethical, regulatory, and research considerations associated with web-based qualitative research. In an era of technology ubiquity and dependence, researchers should consider web-based focus groups a potential qualitative research tool, especially when working with youth.

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