scholarly journals Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Abril Gijsbers ◽  
Yue Zhang ◽  
Ye Gao ◽  
Peter J. Peters ◽  
Raimond B. G. Ravelli
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Crystal Structures ◽  
Quality Standard ◽  
High Yield ◽  
Expression And Purification ◽  
Electron Microscopes ◽  
Yield And Purity ◽  
Purification Protocol ◽  
Protein Part

The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Å resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164–181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.

Transmission electron microscope studies in special ceramics

1978 ◽  
Vol 36 (1) ◽  
pp. 268-269
Author(s):  
A. Zangvil ◽  
L.J. Gauckler ◽  
G. Schneider ◽  
M. Rühle
Keyword(s):  
Phase Diagrams ◽  
Crystal Structures ◽  
Thin Foil ◽  
Limited Extent ◽  
Complex Materials ◽  
X Ray Diffraction ◽  
X Ray ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
Lattice Resolution

The use of high temperature special ceramics which are usually complex materials based on oxides, nitrides, carbides and borides of silicon and aluminum, is critically dependent on their thermomechanical and other physical properties. The investigations of the phase diagrams, crystal structures and microstructural features are essential for better understanding of the macro-properties. Phase diagrams and crystal structures have been studied mainly by X-ray diffraction (XRD). Transmission electron microscopy (TEM) has contributed to this field to a very limited extent; it has been used more extensively in the study of microstructure, phase transformations and lattice defects. Often only TEM can give solutions to numerous problems in the above fields, since the various phases exist in extremely fine grains and subgrain structures; single crystals of appreciable size are often not available. Examples with some of our experimental results from two multicomponent systems are presented here. The standard ion thinning technique was used for the preparation of thin foil samples, which were then investigated with JEOL 200A and Siemens ELMISKOP 102 (for the lattice resolution work) electron microscopes.

scholarly journals Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9

2021 ◽  
Vol 11 (10) ◽  
pp. 4659
Author(s):  
Eun-Jung Kim ◽  
Gyu-Min Im ◽  
Chang-Soo Lee ◽  
Yun-Gon Kim ◽  
Byoung Joon Ko ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Calcium Binding ◽  
Nucleotide Sequences ◽  
Antibody Fragments ◽  
High Yield ◽  
Antigen Binding ◽  
Hybridoma Cell Culture ◽  
Inflammatory Protein ◽  
Inflammatory Processes ◽  
Yield And Purity

The calcium-binding protein S100A9 regulates inflammatory processes and the immune response. It is overexpressed in a variety of inflammatory and oncologic conditions. In this study, we produced a recombinant human S100A9 (hS100A9) antigen with high yield and purity and used it to generate a hybridoma cell culture-based monoclonal anti-hS100A9 antibody. We selected five anti-hS100A9 antibodies from cell supernatants that showed high antigen binding efficiency and identified the nucleotide sequences of three antibodies: two with high effective concentration values and one with the lowest value. The antigen and antibody development procedures described herein are useful for producing large amounts of monoclonal antibodies against hS100A9 and other antigens of interest. The nucleotide sequences of the anti-hS100A9 monoclonal antibody revealed herein will be helpful in the generation of recombinant antibodies or antibody fragments against hS100A9.

scholarly journals Defined MSC exosome with high yield and purity to improve regenerative activity

2021 ◽  
Vol 12 ◽  
pp. 204173142110086
Author(s):  
Jun Yong Kim ◽  
Won-Kyu Rhim ◽  
Yong-In Yoo ◽  
Da-Seul Kim ◽  
Kyoung-Won Ko ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Medium ◽  
Density Lipoprotein ◽  
High Yield ◽  
Human Umbilical Cord ◽  
Cell Culture Medium ◽  
Human Coronary Artery ◽  
Isolation Methods ◽  
Regenerative Activity ◽  
Yield And Purity

Exosomes derived from mesenchymal stem cells (MSCs) have been studied as vital components of regenerative medicine. Typically, various isolation methods of exosomes from cell culture medium have been developed to increase the isolation yield of exosomes. Moreover, the exosome-depletion process of serum has been considered to result in clinically active and highly purified exosomes from the cell culture medium. Our aim was to compare isolation methods, ultracentrifuge (UC)-based conventional method, and tangential flow filtration (TFF) system-based method for separation with high yield, and the bioactivity of the exosome according to the purity of MSC-derived exosome was determined by the ratio of Fetal bovine serum (FBS)-derived exosome to MSC-derived exosome depending on exosome depletion processes of FBS. The TFF-based isolation yield of exosome derived from human umbilical cord MSC (UCMSC) increased two orders (92.5 times) compared to UC-based isolation method. Moreover, by optimizing the process of depleting FBS-derived exosome, the purity of UCMSC-derived exosome, evaluated using the expression level of MSC exosome surface marker (CD73), was about 15.6 times enhanced and the concentration of low-density lipoprotein-cholesterol (LDL-c), known as impurities resulting from FBS, proved to be negligibly detected. The wound healing and angiogenic effects of highly purified UCMSC-derived exosomes were improved about 23.1% and 71.4%, respectively, with human coronary artery endothelial cells (HCAEC). It suggests that the defined MSC exosome with high yield and purity could increase regenerative activity.

Procedure for isolation of gangliosides in high yield and purity: Simultaneous isolation of neutral glycosphingolipids

1985 ◽  
Vol 148 (1) ◽  
pp. 163-173 ◽  
Author(s):  
Mary C. Byrne ◽  
Michele Sbaschnig-Agler ◽  
Dennis A. Aquino ◽  
Joseph R. Sclafani ◽  
Robert W. Ledeen
Keyword(s):  
High Yield ◽  
Neutral Glycosphingolipids ◽  
Yield And Purity

scholarly journals Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research

2019 ◽  
Vol 8 (11) ◽  
pp. 1995 ◽  
Author(s):  
Moon ◽  
Shin ◽  
Kim ◽  
Lee ◽  
Mankhong ◽  
...  
Keyword(s):  
Human Plasma ◽  
Extracellular Vesicles ◽  
Proteinase K ◽  
High Yield ◽  
Clinical Samples ◽  
Diagnostic Biomarkers ◽  
Micro Rnas ◽  
Biomarker Research ◽  
Protein Rich ◽  
Yield And Purity

Exosome-like extracellular vesicles (ELVs) contain biomolecules that have potential as diagnostic biomarkers, such as proteins, micro-RNAs (miRNAs), and lipids. However, it is difficult to enrich ELVs consistently with high yield and purity from clinical samples, which hampers the development of ELV biomarkers. This is particularly true for miRNAs in protein-rich plasma. Hence, we modified ELV isolation protocols of three commercially available polymer-precipitation-based kits using proteinase K (PK) treatment to quantify ELV-associated miRNAs in human plasma. We compared the yield, purity, and characteristics of enriched plasma ELVs, and measured the relative quantity of three selected miRNAs (miR-30c, miR-126, and miR-192) in ELVs using six human plasma samples. Compared with the original protocols, we demonstrated that ELVs can be isolated with PK treatment with high purity (i.e., lack of non-exosomal proteins and homogeneous size of vesicles) and yield (i.e., abundancy of exosomal markers), which were dependent on kits. Using the kit with the highest purity and yield with PK treatment, we successfully quantified ELV miRNAs (levels of 45%–65% in total plasma) with acceptable variability. Collectively, ELV enrichment using the modified easy-to-use method appears suitable for the analysis of miRNAs, although its clinical applicability needs to be confirmed in larger clinical studies.

scholarly journals Crystal structures of Mycobacterium tuberculosis S‐adenosyl‐L‐homocysteine hydrolase in ternary complex with substrate and inhibitors

2008 ◽  
Vol 17 (12) ◽  
pp. 2134-2144 ◽  
Author(s):  
Manchi C.M. Reddy ◽  
Gokulan Kuppan ◽  
Nishant D. Shetty ◽  
Joshua L. Owen ◽  
Thomas R. Ioerger ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Crystal Structures ◽  
Ternary Complex
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