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Methods in Molecular Biology (Clifton,... 2024Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the...
Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.
Topics: Plant Leaves; Plant Proteins; Solubility
PubMed: 38649582
DOI: 10.1007/978-1-0716-3790-6_20 -
Methods in Molecular Biology (Clifton,... 2024The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the...
The importance of humoral immunity to fungal infections remains to be elucidated. In cryptococcosis, patients that fail to generate antibodies against antigens of the fungus Cryptococcus neoformans are more susceptible to the disease, demonstrating the importance of these molecules to the antifungal immune response. Historically, antibodies against C. neoformans have been applied in diagnosis, therapeutics, and as important research tools to elucidate fungal biology. Throughout the process of generating monoclonal antibodies (mAbs) from a single B-cell clone and targeting a single epitope, several immunization steps might be required for the detection of responsive antibodies to the antigen of interest in the serum. This complex mixture of antibodies comprises the polyclonal antibodies. To obtain mAbs, B-lymphocytes are harvested (from spleen or peripheral blood) and fused with tumor myeloma cells, to generate hybridomas that are individually cloned and specifically screened for mAb production. In this chapter, we describe all the necessary steps, from the immunization to polyclonal antibody harvesting, hybridoma generation, and mAb production and purification. Additionally, we discuss new cutting-edge approaches for generating interspecies mAbs, such as humanized mAbs, or for similar species in distinct host backgrounds.
Topics: Cryptococcus neoformans; Antibodies, Monoclonal; Animals; Humans; Hybridomas; Antibodies, Fungal; Mice; B-Lymphocytes; Cryptococcosis; Antigens, Fungal; Immunization
PubMed: 38758326
DOI: 10.1007/978-1-0716-3722-7_20 -
Protein Expression and Purification Jul 2024Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries...
Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus A41L gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (K) being 1.22 × 10 M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic β-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.
Topics: Humans; Viral Proteins; Crystallography, X-Ray; Animals; Interleukin-8; Gene Expression; Sf9 Cells; Recombinant Proteins; Yatapoxvirus
PubMed: 38588871
DOI: 10.1016/j.pep.2024.106480 -
Viruses Mar 2024As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including...
As a mosquito-borne flavivirus, Zika virus (ZIKV) has been identified as a global health threat. The virus has been linked to severe congenital disabilities, including microcephaly and other congenital malformations, resulting in fatal intrauterine death. Therefore, developing sensitive and specific methods for the early detection and accurate diagnosis of the ZIKV is essential for controlling its spread and mitigating its impact on public health. Herein, we set up a novel nucleic acid detection system based on Argonaute (Ago)-mediated nucleic acid detection, targeting the non-structural protein 5 (NS5) region of the ZIKV genome (abbreviated ZIKV-PAND). Without preamplification with the polymerase chain reaction (PCR), the minimum detection concentration (MDC) of ZIKV-PAND was about 10 nM. When introducing an amplification step, the MDC can be dramatically decreased to the aM level (8.3 aM), which is comparable to qRT-PCR assay (1.6 aM). In addition, the diagnostic findings from the analysis of simulated clinical samples or Zika virus samples using ZIKV-PAND show a complete agreement of 100% with qRT-PCR assays. This correlation can aid in the implementation of molecular testing for clinical diagnoses and the investigation of ZIKV infection on an epidemiological scale.
Topics: Zika Virus; Zika Virus Infection; Humans; Viral Nonstructural Proteins; Pyrococcus furiosus; Argonaute Proteins; Sensitivity and Specificity; RNA, Viral; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Genome, Viral
PubMed: 38675882
DOI: 10.3390/v16040539 -
Methods in Molecular Biology (Clifton,... 2024N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They...
N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form the tetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in the eukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.
Topics: Receptors, N-Methyl-D-Aspartate; Animals; Chromatography, Gel; Baculoviridae; Chromatography, Affinity; Humans; Recombinant Proteins; Gene Expression; Sf9 Cells
PubMed: 38727900
DOI: 10.1007/978-1-0716-3830-9_2 -
Critical Reviews in Food Science and... Jun 2024Edible insects are accepted as food and feed ingredients in many parts of the world. Insects account for more than 80% of animal kingdom providing rich biodiversity of... (Review)
Review
Edible insects are accepted as food and feed ingredients in many parts of the world. Insects account for more than 80% of animal kingdom providing rich biodiversity of protein and lipid profiles compared to conventional livestock. Insect biomasses contain an average of 35-62% protein, 3-57% lipid, and 3-12% chitin, and their nutritional values are widely recognized due to their presence, including minerals, and vitamins. While whole insects are consumed as eggs, larvae, pupae, or adults, there has been a recent uptick in interest to use fractions, e.g., protein, lipid, and chitin, as food and feed ingredients. To utilize these fractions in various food and feed preparations, a deeper understanding of the physicochemical as well as functional properties of the ingredients is required, which are generally impacted by extraction and preparation processes. Thus, the methods of extraction/purification are important to preserve the quality and functional properties of these ingredients. This paper discusses the extraction methods for insect protein, lipid, and chitin, their functional properties, and potential applications in food and feed applications.
Topics: Chitin; Animals; Lipids; Insecta; Insect Proteins; Nutritive Value; Edible Insects; Animal Feed
PubMed: 36691837
DOI: 10.1080/10408398.2023.2168620 -
Nucleic Acids Research Aug 2023Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently...
Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently retrieved by needle shearing or heating during RNA extraction. Semi-extractable RNAs are promising resources for understanding RNA-centric phase separation. However, limited assessments have been performed to systematically identify and characterize semi-extractable RNAs. In this study, 1074 semi-extractable RNAs, including ASAP1, DANT2, EXT1, FTX, IGF1R, LIMS1, NEAT1, PHF21A, PVT1, SCMH1, STRG.3024.1, TBL1X, TCF7L2, TVP23C-CDRT4, UBE2E2, ZCCHC7, ZFAND3 and ZSWIM6, which exhibited consistent semi-extractability were identified across five human cell lines. By integrating publicly available datasets, we found that semi-extractable RNAs tend to be distributed in the nuclear compartments but are dissociated from the chromatin. Long and repeat-containing semi-extractable RNAs act as hubs to provide global RNA-RNA interactions. Semi-extractable RNAs were divided into four groups based on their k-mer content. The NEAT1 group preferred to interact with paraspeckle proteins, such as FUS and NONO, implying that RNAs in this group are potential candidates of architectural RNAs that constitute nuclear bodies.
Topics: Humans; Cell Line; Cell Nucleus; Chromatin; DNA-Binding Proteins; RNA; RNA, Long Noncoding
PubMed: 37463833
DOI: 10.1093/nar/gkad567 -
Protein Expression and Purification Sep 2024Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for...
Nanobodies are single-variable domain antibodies with excellent properties, which are evolving as versatile tools to guide cognate antigens in vitro and in vivo for biological research, diagnosis, and treatment. Given their simple structure, nanobodies are readily produced in multiple systems. However, selecting an appropriate expression system is crucial because different conditions might cause proteins to produce different folds or post-translational modifications (PTMs), and these differences often result in different functions. At present, the strategies of PTMs are rarely reported. The GFP nanobody can specifically target the GFP protein. Here, we engineered a GFP nanobody fused with 6 × His tag and Fc tag, respectively, and expressed in bacteria and mammalian cells. The 6 × His-GFP-nanobody was produced from Escherichia coli at high yields and the pull-down assay indicated that it can precipitate the GFP protein. Meanwhile, the Fc-GFP-nanobody can be expressed in HEK293T cells, and the co-immunoprecipitation experiment can trace and target the GFP-tagged protein in vivo. Furthermore, some different PTMs in antigen-binding regions have been identified after using mass spectrometry (MS) to analyze the GFP nanobodies, which are expressed in prokaryotes and eukaryotes. In this study, a GFP nanobody was designed, and its binding ability was verified by using the eukaryotic and prokaryotic protein expression systems. In addition, this GFP nanobody was transformed into a useful instrument for more in-depth functional investigations of GFP fusion proteins. MS was further used to explore the reason for the difference in binding ability, providing a novel perspective for the study of GFP nanobodies and protein expression purification.
Topics: Humans; Green Fluorescent Proteins; Single-Domain Antibodies; HEK293 Cells; Protein Processing, Post-Translational; Escherichia coli; Recombinant Fusion Proteins; Protein Engineering; Gene Expression
PubMed: 38782081
DOI: 10.1016/j.pep.2024.106501 -
Food Research International (Ottawa,... Dec 2023Collagen peptides play an important role in the increasing use of collagen peptides as dietary supplements in food and beverages and as bioactive ingredients in...
Collagen peptides play an important role in the increasing use of collagen peptides as dietary supplements in food and beverages and as bioactive ingredients in cosmetics, healthcare, and pharmaceuticals. Collagenase enzymatically cleaves gelatin to produce collagen polypeptides. However, the enzymatic activity of collagenase is very low (25900 U) and does not allow for adequate enzymatic digestion. Therefore, proteases are used to assist in enzymatic digestion. Porcine gelatin, bovine gelatin, and fish protein gum were enzymatically digested, and the content of collagen peptides in the enzymatically digested lyophilized powder was identified by high-performance liquid chromatography and mass spectrometry, and then the content of the desired collagen peptides was increased by isolation and purification, and the result of the determination was that the content of collagen peptides was the highest after enzymatic digestion and isolation and purification with the use of porcine gelatin as the raw material, and the content of the collagen peptides was about 45.47%. β-nicotinamide mononucleotide (NMN) was mixed with the prepared samples to determine its antioxidant properties and ability to promote the growth of human dermal fibroblasts. The results showed that the antioxidant capacity was enhanced with the increase of collagen polypeptide content, and NMN could promote the scavenging of DPPH• and •OH free radicals by collagen polypeptides. The ability to promote the growth of human dermal fibroblasts was enhanced with the increase of collagen polypeptide content. This paper aimed to prepare a high content of collagen polypeptides from three raw materials, porcine gelatin, bovine gelatin, and fish protein gum, and further to determine the biological activities.
Topics: Animals; Cattle; Humans; Gelatin; Antioxidants; Peptides; Collagen; Collagenases; Fish Proteins
PubMed: 37986438
DOI: 10.1016/j.foodres.2023.113561 -
Clinical Microbiology and Infection :... Nov 2023To investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae...
OBJECTIVE
To investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates.
METHODS
The mechanism of high-level tigecycline resistance in CRKP mediated by a tet(A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet(A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR.
RESULTS
A high rate (62.1%, 998/1607) of tet(A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet(A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the ΔtnpA homologous sequence of Tn1721 supports the amplification of the region that harbours the tet(A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet(A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present.
CONCLUSION
The presence of a tigecycline resistance-mediating tet(A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently.
Topics: Tigecycline; Klebsiella pneumoniae; Humans; Anti-Bacterial Agents; Klebsiella Infections; Microbial Sensitivity Tests; Bacterial Proteins; China; Whole Genome Sequencing; Carbapenems; Drug Resistance, Bacterial; Carbapenem-Resistant Enterobacteriaceae; Gene Amplification; Plasmids; Antiporters
PubMed: 37549732
DOI: 10.1016/j.cmi.2023.07.030