Browse
Submit Data
Databases
API
Help

Dataset Information

9 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Titer measurement of HIV-1 envelope trimeric glycoprotein in cell culture media by a new tandem ion exchange and size exclusion chromatography (IEC-SEC) method.

ABSTRACT: An efficient and specific liquid chromatography (LC)-based assay was developed to monitor the production of recombinant HIV-1 trimeric envelope glycoprotein (HIV Env trimer), a candidate vaccine for HIV-1 infection, in cell culture media to support scale-up process development. In this method, titer measurement was achieved by coupling a weak anion exchange chromatography (IEC) column with a size exclusion chromatography (SEC) column. This assay was specific, accurate, precise, and has been qualified for its intended purpose, with a limit of quantification (LOQ) of 10 µg/mL. This tandem separation strategy offered a reliable and timely analytical support to directly monitor the titer of HIV Env trimer during cell growth, without any extra sample purification steps.

SUBMITTER: Yang G

PROVIDER: S-EPMC8130148 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

dichotomic size-exclusion chromatography（SEC）

Project description:directDIA proteomics raw data of plasma extracellular vesicles isolated by SEC and UC

2021-05-17 | PXD026034 |

Insights into the trimeric HIV-1 envelope glycoprotein structure.

Project description:The HIV-1 envelope glycoprotein (Env) trimer is responsible for receptor recognition and viral fusion with CD4(+) T cells, and is the sole target for neutralizing antibodies. Thus, understanding its molecular architecture is of significant interest. However, the Env trimer has proved to be a challenging target for 3D structure determination. Recent electron microscopy (EM) and X-ray structures have at last enabled us to decipher the structural complexity and unique features of the Env trimer, and how it is recognized by an ever-expanding arsenal of potent broadly neutralizing antibodies. We describe our current knowledge of the Env trimer structure in the context of exciting recent developments in the identification and characterization of HIV broadly neutralizing antibodies.

| S-EPMC4310573 | biostudies-literature

Structural mechanism of trimeric HIV-1 envelope glycoprotein activation.

Project description:HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic. Binding of trimeric HIV-1 BaL Env to either soluble CD4 or 17b alone, is sufficient to trigger formation of the open quaternary conformation of Env. In contrast, VRC01 locks Env in the closed state, while b12 binding requires a partial opening in the quaternary structure of trimeric Env. Our results show that, despite general similarities in regions of the HIV-1 gp120 polypeptide that contact CD4, VRC01, VRC03 and b12, there are important differences in quaternary structures of the complexes these ligands form on native trimeric Env, and potentially explain differences in the neutralizing breadth and potency of antibodies with similar specificities. From cryo-electron microscopic analysis at ∼9 Å resolution of a cleaved, soluble version of trimeric Env, we show that a structural signature of the open Env conformation is a three-helix motif composed of α-helical segments derived from highly conserved, non-glycosylated N-terminal regions of the gp41 trimer. The three N-terminal gp41 helices in this novel, activated Env conformation are held apart by their interactions with the rest of Env, and are less compactly packed than in the post-fusion, six-helix bundle state. These findings suggest a new structural template for designing immunogens that can elicit antibodies targeting HIV at a vulnerable, pre-entry stage.

| S-EPMC3395603 | biostudies-literature

Novel trimeric human cytomegalovirus glycoprotein B elicits a high-titer neutralizing antibody response.

Project description:Human cytomegalovirus (HCMV) is a major cause of disability in congenitally infected infants and in the immunosuppressed. There is currently no licensed prophylactic HCMV vaccine. The HCMV envelope glycoprotein B (gB) is considered a major vaccine target antigen based on its critical role in mediating viral-host cell fusion and thus viral entry. The natural conformation of HCMV gB within the viral envelope is a trimer, but there has been no reported success in producing a recombinant trimeric gB suitable for vaccine use. Phase II clinical trials of a monomeric recombinant gB protein demonstrated 50% efficacy in preventing HCMV infection in seronegative women of reproductive age, and in reducing viremia in solid organ transplantation recipients. We now report the production of a uniformly trimeric recombinant HCMV gB protein in Chinese ovary cells, as demonstrated by Western blot analysis under modified non-reducing conditions and size exclusion chromatography with multi-angle scattering. Immunization of mice with trimeric HCMV gB induced up to 11-fold higher serum titers of total gB-specific IgG relative to monomeric HCMV gB using Alum + CpG as adjuvants. Further, trimeric HCMV gB elicited 50-fold higher complement-independent and 20-fold higher complement-dependent HCMV neutralizing titers compared to monomeric HCMV gB using the fibroblast cell line, MRC-5, and up to 6-fold higher complement-independent and -dependent HCMV neutralizing titers using the epithelial cell line, ARPE-19. The markedly enhanced HCMV neutralizing activity in response to trimeric HCMV gB was also observed using an additional four distinct clinical HCMV isolates. These data support a role for trimeric HCMV gB as an important component for clinical testing of a prophylactic HCMV vaccine.

| S-EPMC6556890 | biostudies-literature

Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy.

Project description:The activation of trimeric HIV-1 envelope glycoprotein (Env) by its binding to the cell-surface receptor CD4 and co-receptors (CCR5 or CXCR4) represents the first of a series of events that lead to fusion between viral and target-cell membranes. Here, we present the cryo-EM structure, at subnanometer resolution (~6 Å at 0.143 FSC), of the 'closed', prefusion state of trimeric HIV-1 Env complexed to the broadly neutralizing antibody VRC03. We show that three gp41 helices at the core of the trimer serve as an anchor around which the rest of Env is reorganized upon activation to the 'open' quaternary conformation. The architecture of trimeric HIV-1 Env in the prefusion state and in the activated intermediate state resembles the corresponding states of influenza hemagglutinin trimers, thus providing direct evidence for the similarity in entry mechanisms used by HIV-1, influenza and related enveloped viruses.

| S-EPMC3917492 | biostudies-literature

Ultra-high performance liquid chromatography-size exclusion chromatography (UPLC-SEC) as an efficient tool for the rapid and highly informative characterisation of biopolymers.

Project description:Starch has a complex molecular structure, with properties dependent on the relative chain lengths and branching structure of its constituent molecules, which varies due to polymorphisms in starch biosynthetic genes, as well as environmental factors. Here we present the application of ultra-high performance size exclusion chromatography to the separation of starch chains from plant seeds. Several methods, have been used to analyse chain length distributions in starch, all with limitations in terms of analysis time, sample preparation and molecular weight range. Here we demonstrate that chain length distributions can be obtained with dramatically reduced analysis time using ultra-high performance size exclusion chromatography. The method may also show improvements in resolution of some fine structural features. Understanding links between starch fine structure and biosynthetic genes will allow bioengineering of starches with tailored properties. This technique may have application to the size separation and resolution of a range of biopolymers of value to the food, drink and pharmaceutical industries.

| S-EPMC6030444 | biostudies-literature

Fluorophore Absorption Size Exclusion Chromatography (FA-SEC): An Alternative Method for High-Throughput Detergent Screening of Membrane Proteins.

Project description:Membrane proteins play key roles in many fundamental functions in cells including ATP synthesis, ion and molecule transporter, cell signalling and enzymatic reactions, accounting for ~30% genes of whole genomes. However, the hydrophobic nature of membrane proteins frequently hampers the progress of structure determination. Detergent screening is the critical step in obtaining stable detergent-solubilized membrane proteins and well-diffracting protein crystals. Fluorescence Detection Size Exclusion Chromatography (FSEC) has been developed to monitor the extraction efficiency and monodispersity of membrane proteins in detergent micelles. By tracing the FSEC profiles of GFP-fused membrane proteins, this method significantly enhances the throughput of detergent screening. However, current methods to acquire FSEC profiles require either an in-line fluorescence detector with the SEC equipment or an off-line spectrofluorometer microplate reader. Here, we introduce an alternative method detecting the absorption of GFP (FA-SEC) at 485 nm, thus making this methodology possible on conventional SEC equipment through the in-line absorbance spectrometer. The results demonstrate that absorption is in great correlation with fluorescence of GFP. The comparably weaker absorption signal can be improved by using a longer path-length flow cell. The FA-SEC profiles were congruent with the ones plotted by FSEC, suggesting FA-SEC could be a comparable and economical setup for detergent screening of membrane proteins.

| S-EPMC4917255 | biostudies-literature

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein.

Project description:The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs) that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664) maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

| S-EPMC4805854 | biostudies-literature

Global profiling of the RNA and protein complexes of Escherichia coli by size exclusion chromatography followed by RNA sequencing and mass spectrometry (SEC-seq)

Project description:New methods for the global identification of RNA-protein interactions have led to greater recognition of the abundance and importance of RNA-binding proteins (RBPs) in bacteria. Here, we expand this tool kit by developing SEC-seq, a method based on a similar concept as the established Grad-seq approach. In Grad-seq, cellular RNA and protein complexes of a bacterium of interest are separated in a glycerol gradient, followed by high-throughput RNA-sequencing and mass spectrometry analyses of individual gradient fractions. New RNA-protein complexes are predicted based on the similarity of their elution profiles. In SEC-seq, we have replaced the glycerol gradient with separation by size exclusion chromatography, which shortens operation times and offers greater potential for automation. Applying SEC-seq to Escherichia coli, we find that the method provides a higher resolution than Grad-seq in the lower molecular weight range up to ~500 kDa. This is illustrated by the ability of SEC-seq to resolve two distinct, but similarly sized complexes of the global translational repressor CsrA with either of its antagonistic small RNAs, CsrB and CsrC. We also characterized changes in the SEC-seq profiles of the small RNA MicA upon deletion of its RNA chaperones Hfq and ProQ and investigated the redistribution of these two proteins upon RNase treatment. Overall, we demonstrate that SEC-seq is a tractable and reproducible method for the global profiling of bacterial RNA-protein complexes that offers the potential to discover yet-unrecognized associations between bacterial RNAs and proteins.

2022-10-27 | GSE212408 | GEO

Pre-fusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy

Project description: Not available

| EMPIAR-10004 | biostudies-other