Abstract 437: Optimizing a Prokaryotic Cell Free Expression System for the Generation of synthetic ApoB-Containing Lipoprotein Particles

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Humra Athar ◽  
Zhenghui G Jiang ◽  
Christopher J McKnight
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Low Density Lipoproteins ◽  
Free Expression ◽  
Low Density ◽  
Prokaryotic Cell ◽  
Lipoprotein Particles ◽  
Cell Free Expression System

High serum levels of low density lipoproteins (LDL) is associated with increased risk of atherosclerosis. Apolipoprotein B (ApoB) is required for the assembly and secretion of chylomicrons and very low density lipoproteins (VLDL), the precursor of low density lipoproteins (LDL). Despite its clinical significance, the mechanism of the assembly of these ApoB containing lipoproteins is poorly understood. The assembly process is an interplay of several key components including but not limited to nascent ApoB, lipids, ER resident chaperones and importantly, microsomal triglyceride transfer protein (MTP). In the current study, we are trying to understand several unanswered questions in the mechanism of the lipoprotein assembly. We have used a novel prokaryotic cell-free expression system and lipids mimicking the ER membrane to produce particles that represent the early dense initiation particles formed in the ER. After optimizing several different conditions, we were able to make “synthetic” lipoproteins by cotranslational expression of constructs from the first 22% of ApoB tagged with a 6-histidine tag at the C-terminus (ApoB 22-His) with small unilamellar phosphatidylcholine (PC) vesicles and phosphatidylcholine:triolein (PC:TO) emulsions. After cotranslational interaction with lipids, these constructs migrate to a lower density in potassium bromide (KBr) density gradient centrifugation. Here we report a new ApoB 22 construct with a FLAG tag at the N-terminus in addition to the C-terminal His tag. The construct makes significant amount of soluble protein that is soluble in the cell free reaction. The two N- and C-terminal tags allow us to purify full length construct from any truncation products. In addition, the dual-tag approach will allow us to purify the synthetic lipoproteins directly from the cell free system, and thereby avoid the requirement for KBr density gradient centrifugation. This new strategy will provide far more efficient generation and purification of synthetic ApoB containing lipoprotein particles.

scholarly journals Low-Density Granulocyte Contamination From Peripheral Blood Mononuclear Cells of Patients With Sepsis and How to Remove It – A Technical Report

2021 ◽  
Vol 12 ◽  
Author(s):  
Judith Schenz ◽  
Manuel Obermaier ◽  
Sandra Uhle ◽  
Markus Alexander Weigand ◽  
Florian Uhle
Keyword(s):  
Healthy Volunteers ◽  
Density Gradient ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15+ cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15+ cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

scholarly journals Plasma lipoprotein and apolipoprotein distribution as a function of density in the rainbow trout (Salmo gairdneri)

1987 ◽  
Vol 246 (2) ◽  
pp. 425-429 ◽  
Author(s):  
P J Babin
Keyword(s):  
Plasma Proteins ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density Lipoproteins ◽  
Plasma Lipoproteins ◽  
Single Type ◽  
Salmo Gairdneri ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Intermediate Density

I have previously described [Babin (1987) J. Biol. Chem. 262, 4290-4296] the apolipoprotein composition of the major classes of trout plasma lipoproteins. The present work describes the use of an isopycnic density gradient centrifugation procedure and sequential flotation ultracentrifugation to show: (1) the presence of intermediate density lipoproteins (IDL) in the plasma, between 1.015 and 1.040 g/ml; (2) the existence of a single type of Mr 240,000 apoB-like in the low density lipoproteins (LDL, 1.040 less than p less than 1.085 g/ml); (3) the presence of apoA-I-like (Mr 25,000) in the densest LDL; (4) the adequacy of 1.085 g/ml as a cutoff between the LDL and high density lipoproteins (HDL); (5) the accumulation of Mr 55,000 and 76,000 apolipoproteins and apoA-like apolipoproteins in the 1.21 g/ml infranatant. The fractionation of trout lipoprotein spectrum thus furnishes the distribution of the different lipoprotein classes and leads to the description of the constituent apolipoproteins, which account for about 36% of circulating plasma proteins in this species.

The Presence of Glycogen Synthase in Preparations of Platelet Plasma Membranes

1973 ◽  
Vol 30 (02) ◽  
pp. 307-314 ◽  
Author(s):  
J. H Greenberg ◽  
A. P Fletcher ◽  
G. A Jamieson
Keyword(s):  
Density Gradient ◽  
Plasma Membranes ◽  
Density Gradient Centrifugation ◽  
Glycogen Synthase ◽  
Gradient Centrifugation ◽  
Low Density ◽  
Trichloracetic Acid ◽  
Continuous Density ◽  
Platelet Membranes ◽  
Glycogen Synthase Activity

SummaryGlycogen synthase and glycogen (63 ± 35 μg/unit) have been identified in partially-purified preparations of platelet membranes isolated by density-step centrifugation following glycerol-lysis; this combination is responsible for the endogenous (collagen-independent) activity measured during the assay of collagen: glucosyltransferase. Glycogen synthesized in this way may be complexed with protein since it is precip-itable with trichloracetic acid. High and low density membranes prepared by continuous density gradient centrifugation are devoid of glycogen synthase activity.

scholarly journals Proof-of-concept for the yadokari nature: a capsidless replicase-encoding but replication-dependent (+)ssRNA virus hosted by an unrelated dsRNA virus

2021 ◽  
Author(s):  
Subha Das ◽  
Md Mahfuz Alam ◽  
Rui Zhang ◽  
Sakae Hisano ◽  
Nobuhiro Suzuki
Keyword(s):  
Density Gradient ◽  
Reverse Genetics ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Expression System ◽  
Cesium Chloride ◽  
Dsrna Virus ◽  
Equilibrium Density ◽  
Proof Of Concept ◽  
Ssrna Virus

We have previously proposed a new virus lifestyle or yadokari/yadonushi nature exhibited by a positive-sense ssRNA virus, yadokari virus 1 (YkV1), and an unrelated dsRNA virus, yadonushi virus 1 (YnV1) in a phytopathogenic ascomycete, Rosellinia necatrix . We have proposed that YkV1 diverts the YnV1 capsid to trans-encapsidate YkV1 RNA and RNA-dependent RNA polymerase (RdRp) and replicate in the heterocapsid. However, it remains uncertain whether YkV1 replicates using its own RdRp, and whether YnV1 capsid co-packages both YkV1 and YnV1 components. To address these questions, we first took advantage of the reverse genetics tools available for YkV1. Mutations in the GDD RdRp motif, one of the two identifiable functional motifs on the YkV1 polyprotein, abolished its replication competency. Mutations were also introduced in the conserved 2A-like peptide motif, hypothesized to cleave the YkV1 polyprotein co-translationally. Interestingly, the replication proficiency of YkV1 mutants in the host fungus agreed with the cleavage activity of the 2A-like peptide tested using a baculovirus expression system. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with a subset of YnV1 capsid solely packaging YkV1 dsRNA and RdRp. These results provide proof-of-concept that a capsidless (+)ssRNA virus is hosted by an unrelated dsRNA virus. Importance Viruses typically encode their own capsids that encase their genomes. However, a capsidless (+)ssRNA virus, YkV1, depends on an unrelated dsRNA virus, YnV1, for encapsidation and replication. We have previously shown that YkV1 highjacks the capsid of YnV1 for trans-encapsidation of its own RNA and RdRp. YkV1 was hypothesized to divert the hetero-capsid as the replication site, as is commonly observed for dsRNA viruses. Herein, mutational analyses showed that the RdRp and 2A-like domains on the YkV1 polyprotein are important for its replication. The active RdRp must be cleaved by a 2A-like peptide from the C-proximal protein. Cesium chloride equilibrium density gradient centrifugation allowed for the separation of particles, with YnV1 capsid solely packaging YkV1 dsRNA and RdRp. This study provides proof-of-concept of a virus neo-lifestyle where a (+)ssRNA virus snatches capsids from an unrelated dsRNA virus to replicate with its own RdRp, thereby mimicking the typical dsRNA virus lifestyle.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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