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Protein Expression and Purification Jul 2024The bacterial Efe system functions as an importer of free Fe into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding...
The bacterial Efe system functions as an importer of free Fe into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.
Topics: Escherichia coli; Crystallography, X-Ray; Escherichia coli Proteins; Iron; Gene Expression; Recombinant Proteins; Iron-Binding Proteins
PubMed: 38657915
DOI: 10.1016/j.pep.2024.106487 -
Phytochemistry Aug 2023A bioactivity-guided isolation from the aerial parts of Phyllanthus rheophyticus obtained 17 undescribed ent-cleistanthane-type diterpenoids, namely phyllarheophols A-Q,...
A bioactivity-guided isolation from the aerial parts of Phyllanthus rheophyticus obtained 17 undescribed ent-cleistanthane-type diterpenoids, namely phyllarheophols A-Q, as well as 12 known analogs. Their structures were characterized by a combination of spectroscopic data interpretation, single-crystal X-ray diffraction and ECD analysis. The anti-inflammatory activities of these compounds were evaluated by measuring their inhibitory effects on NO production in LPS-stimulated RAW264.7 macrophages, and their preliminary structure-activity relationships were also discussed. Further study showed that promising compounds phyllarheophol D and phyacioid B significantly suppressed the expressions of cytokines and nitric oxide synthase through the NF-κB signaling pathway.
Topics: Anti-Inflammatory Agents; Diterpenes; Lipopolysaccharides; Macrophages; Molecular Structure; Nitric Oxide; Phyllanthus; Structure-Activity Relationship; NF-kappa B; Plant Components, Aerial; Tumor Necrosis Factor-alpha; Interleukin-6; Nitric Oxide Synthase Type II; RAW 264.7 Cells; Animals; Mice
PubMed: 37182686
DOI: 10.1016/j.phytochem.2023.113723 -
Journal of Chromatography. A Dec 2023Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide... (Review)
Review
Field-flow fractionation (FFF) with its several variants, has developed into a mature methodology. The scope of the FFF investigations has expanded, covering both a wide range of basic studies and especially a wide range of analytical applications. Special attention of this review is given to the achievements of FFF with reference to recent applications in the fractionation, isolation, and purification of biomacromolecules, and from which especially those of (in alphabetical order) bacteria, cells, extracellular vesicles, liposomes, lipoproteins, nucleic acids, and viruses and virus-like particles. In evaluating the major approaches and trends demonstrated since 2012, the most significant biomacromolecule applications are compiled in tables. It is also evident that asymmetrical flow field-flow fractionation is by far the most dominant technique in the studies. The industry has also shown current interest in FFF and adopted it in some sophisticated fields. FFF, in combination with appropriate detectors, handles biomacromolecules in open channel in a gentle way due to the lack of shear forces and unwanted interactions caused by the stationary phase present in chromatography. In addition, in isolation and purification of biomacromolecules quite high yields can be achieved under optimal conditions.
Topics: Chemical Fractionation; Fractionation, Field Flow; Lipoproteins; Chromatography; Liposomes
PubMed: 37944435
DOI: 10.1016/j.chroma.2023.464492 -
International Journal of Biological... May 2024Rice protein is highly nutritious and easy to digest and absorb. Its hydrolyzed peptides have significant effects on lowering blood pressure and cholesterol. First, a... (Review)
Review
Rice protein is highly nutritious and easy to digest and absorb. Its hydrolyzed peptides have significant effects on lowering blood pressure and cholesterol. First, a detailed and comprehensive explanation of rice protein extraction methods was given, and it was found that the combination of enzymatic and physical methods could improve the extraction rate of rice protein, but it was only suitable for laboratory studies. Second, the methods for improving the functional properties of rice protein were introduced, including physical modification, chemical modification, and enzymatic modification. Enzymatic modification of the solubility of rice protein to improve its functional properties has certain limitations due to the low degree of hydrolysis, the long time required, the low utilization of the enzyme, and the possible undesirable taste of the product. Finally, the development and utilization of rice protein was summarized and the future research direction was suggested. This paper lists the advantages and disadvantages of various extraction techniques, points out the shortcomings of existing extraction techniques, aims to fill the gap in the field of rice protein extraction, and then provides a possible improvement method for the extraction and development of rice protein in the future.
Topics: Oryza; Plant Proteins; Solubility; Hydrolysis; Chemical Fractionation
PubMed: 38643916
DOI: 10.1016/j.ijbiomac.2024.131705 -
STAR Protocols Jun 2024The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein,...
The microbial transcription factor YhaJ responds to 2,4-dinitrotoluene (DNT) derivatives. Here, we describe steps for overexpression and purification of the protein, characterization for the binding of a DNT derivative methylhydroquinone, and crystallization by using a random seeding technique. We then detail procedures for structure determination by employing the crystal-twin resolving processes. This protocol can also be performed using other DNT derivatives. For complete details on the use and execution of this protocol, please refer to Kim et al..
Topics: Crystallization; Dinitrobenzenes; Crystallography, X-Ray; Transcription Factors; Bacterial Proteins
PubMed: 38573865
DOI: 10.1016/j.xpro.2024.102999 -
The Journal of Biological Chemistry Jan 2024Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular...
Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (K = 1.8 μM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.
Topics: Animals; Cattle; Humans; Calreticulin; Fibrinolysin; Plasminogen; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator; Protein Domains; Mutation; Recombinant Proteins; Gene Knockout Techniques; Cell Line, Tumor; Neoplasms
PubMed: 37979915
DOI: 10.1016/j.jbc.2023.105465 -
Applied Spectroscopy Dec 2023Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific...
Protein A affinity chromatography is a key step in isolation of biotherapeutics (BTs) containing fragment crystallizable regions, including monoclonal and bispecific antibodies. Dynamic binding capacity (DBC) analysis assesses how much BT will bind to a protein A column. DBC reduces with column usage, effectively reducing the amount of recovered product over time. Drug regulatory bodies mandate chromatography resin lifetime for BT isolation, through measurement of parameters including DBC, so this feature is carefully monitored in industrial purification pipelines. High-performance affinity chromatography (HPAC) is typically used to assess the concentration of BT, which when loaded to the column results in significant breakthrough of BT in the flowthrough. HPAC gives an accurate assessment of DBC and how this changes over time but only reports on protein concentration, requires calibration for each new BT analyzed, and can only be used offline. Here we utilized Raman spectroscopy and revealed that this approach is at least as effective as both HPAC and ultraviolet chromatogram methods at monitoring DBC of protein A resins. In addition to reporting on protein concentration, the chemical information in the Raman spectra provides information on aggregation status and protein structure, providing extra quality controls to industrial bioprocessing pipelines. In combination with partial least square (PLS) analysis, Raman spectroscopy can be used to determine the DBC of a BT without prior calibration. Here we performed Raman analysis offline in a 96-well plate format, however, it is feasible to perform this inline. This study demonstrates the power of Raman spectroscopy as a significantly improved approach to DBC monitoring in industrial pipelines.
Topics: Spectrum Analysis, Raman; Chromatography, Affinity; Proteins; Staphylococcal Protein A; Calibration
PubMed: 37908083
DOI: 10.1177/00037028231210293 -
Molecules (Basel, Switzerland) Jun 2024Anthocyanins, as the most critical water-soluble pigments in nature, are widely present in roots, stems, leaves, flowers, fruits, and fruit peels. Many studies have... (Review)
Review
Anthocyanins, as the most critical water-soluble pigments in nature, are widely present in roots, stems, leaves, flowers, fruits, and fruit peels. Many studies have indicated that anthocyanins exhibit various biological activities including antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, vision protection, and anti-aging. Hence, anthocyanins are widely used in food, medicine, and cosmetics. The green and efficient extraction and purification of anthocyanins are an important prerequisite for their further development and utilization. However, the poor stability and low bioavailability of anthocyanins limit their application. Protein, one of the three essential nutrients for the human body, has good biocompatibility and biodegradability. Proteins are commonly used in food processing, but their functional properties need to be improved. Notably, anthocyanins can interact with proteins through covalent and non-covalent means during food processing, which can effectively improve the stability of anthocyanins and enhance their bioavailability. Moreover, the interactions between proteins and anthocyanins can also improve the functional characteristics and enhance the nutritional quality of proteins. Hence, this article systematically reviews the extraction and purification methods for anthocyanins. Moreover, this review also systematically summarizes the effect of the interactions between anthocyanins and proteins on the bioavailability of anthocyanins and their impact on protein properties. Furthermore, we also introduce the application of the interaction between anthocyanins and proteins. The findings can provide a theoretical reference for the application of anthocyanins and proteins in food deep processing.
Topics: Anthocyanins; Humans; Proteins; Antioxidants; Biological Availability; Plant Extracts
PubMed: 38930881
DOI: 10.3390/molecules29122815 -
Current Protocols Jun 2024U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes....
U1-70K (snRNP70) serves as an indispensable protein component within the U1 complex, assuming a pivotal role in both constitutive and alternative RNA splicing processes. Notably, U1-70K engages in interactions with SR proteins, instigating the assembly of the spliceosome. This protein undergoes regulation through phosphorylation at multiple sites. Of significant interest, U1-70K has been implicated in Alzheimer's disease, in which it tends to form detergent-insoluble aggregates. Even though it was identified more than three decades ago, our understanding of U1-70K remains notably constrained, primarily due to challenges such as low levels of recombinant expression, susceptibility to protein degradation, and insolubility. In endeavoring to address these limitations, we devised a multifaceted approach encompassing codon optimization, strategic purification, and a solubilization protocol. This methodology has enabled us to achieve a high yield of full-length, soluble U1-70K, paving the way for its comprehensive biophysical and biochemical characterization. Furthermore, we provide a detailed protocol for the preparation of phosphorylated U1-70K. This set of protocols promises to be a valuable resource for scientists exploring the intricate web of U1-70K-related mechanisms in the context of RNA splicing and its implications in neurodegenerative disorders and other disorders and biological processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression and purification of full-length U1-70K from E. coli Support Protocol 1: Making chemically competent BL21 Star pRARE/pBB535 cells Basic Protocol 2: Phosphorylation of full-length U1-70K using SRPK1 Support Protocol 2: Purification of SRPK1 Basic Protocol 3: Expression and purification of U1-70K BAD1 from E. coli Basic Protocol 4: Phosphorylation of U1-70K BAD1 using SRPK1 Basic Protocol 5: Expression and purification of U1-70K BAD2 from E. coli.
Topics: Escherichia coli; Humans; Ribonucleoprotein, U1 Small Nuclear; Phosphorylation; Recombinant Proteins; Gene Expression; Protein Domains
PubMed: 38896106
DOI: 10.1002/cpz1.1059 -
Current Pharmaceutical Biotechnology 2024Chinese hamster ovary cells are the main expression system for recombinant therapeutic proteins. During the production of these proteins, certain host cell proteins are... (Review)
Review
Chinese hamster ovary cells are the main expression system for recombinant therapeutic proteins. During the production of these proteins, certain host cell proteins are secreted, broken down, and released by host cells in the culture along with the proteins of interest. These host cell proteins are often difficult to remove during the downstream purification process, and thus affect the quality, safety, and effectiveness of recombinant protein biopharmaceutical products and increase the production cost of recombinant therapeutic proteins. Therefore, host cell protein production must be reduced as much as possible during the production process and eliminated during purification. This article reviews the harm caused by host cell proteins in the production of recombinant protein drugs using Chinese hamster ovary cell, factors affecting host cell proteins, the monitoring and identification of these proteins, and methods to reduce their type and quantity in the final product.
Topics: Animals; CHO Cells; Cricetulus; Recombinant Proteins; Cricetinae; Humans
PubMed: 37594091
DOI: 10.2174/1389201024666230818112633