scholarly journals Epitope-Independent Purification of Native-Like Envelope Trimers from Diverse HIV-1 Isolates

2016 ◽  
Vol 90 (20) ◽  
pp. 9471-9482 ◽  
Author(s):  
Hans P. Verkerke ◽  
James A. Williams ◽  
Miklos Guttman ◽  
Cassandra A. Simonich ◽  
Yu Liang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Large Scale ◽  
Quaternary Structure ◽  
Vaccine Development ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Lectin Affinity Chromatography ◽  
Native Page ◽  
Antigenic Properties ◽  
Hiv 1

ABSTRACTSoluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers.IMPORTANCEThe production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

scholarly journals The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A

1992 ◽  
Vol 287 (3) ◽  
pp. 979-983 ◽  
Author(s):  
R D Poretz ◽  
R S Yang ◽  
B Canas ◽  
H Lackland ◽  
S Stein ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Human Platelet ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Structural Basis ◽  
Glycan Structure ◽  
Lectin Affinity Chromatography ◽  
Charge Heterogeneity ◽  
Arylsulphatase A ◽  
Polypeptide Structure

Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.

scholarly journals Conformational States of a Soluble, Uncleaved HIV-1 Envelope Trimer

2017 ◽  
Vol 91 (10) ◽  
Author(s):  
Yuhang Liu ◽  
Junhua Pan ◽  
Yongfei Cai ◽  
Nikolaus Grigorieff ◽  
Stephen C. Harrison ◽  
...  
Keyword(s):  
Viral Entry ◽  
Clinical Studies ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Recombinant Form ◽  
Soluble Form ◽  
Compact Structure ◽  
Antigenic Properties ◽  
Hiv 1 ◽  
Gp140 Trimer

ABSTRACT The HIV-1 envelope spike [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers—the uncleaved ectodomains of (gp160)3—from a few selected viral isolates adopt a compact conformation with many antigenic properties of native Env spikes. One is currently being evaluated in a clinical trial. We report here low-resolution (20 Å) electron cryomicroscopy (cryoEM) structures of this gp140 trimer, which adopts two principal conformations, one closed and the other slightly open. The former is indistinguishable at this resolution from those adopted by a stabilized, cleaved trimer (SOSIP) or by a membrane-bound Env trimer with a truncated cytoplasmic tail (EnvΔCT). The latter conformation is closer to a partially open Env trimer than to the fully open conformation induced by CD4. These results show that a stable, uncleaved HIV-1 gp140 trimer has a compact structure close to that of native Env. IMPORTANCE Development of any HIV vaccine with a protein component (for either priming or boosting) requires production of a recombinant form to mimic the trimeric, functional HIV-1 envelope spike in quantities suitable for clinical studies. Our understanding of the envelope structure has depended in part on a cleaved, soluble trimer, known as SOSIP.664, stabilized by several modifications, including an engineered disulfide. This construct, which is difficult to produce in large quantities, has yet to induce better antibody responses than those to other envelope-based immunogens, even in animal models. The uncleaved ectodomain of the envelope protein, called gp140, has also been made as a soluble form to mimic the native Env present on the virion surface. Most HIV-1 gp140 preparations are not stable, however, and have an inhomogeneous conformation. The results presented here show that gp140 preparations from suitable isolates can adopt a compact, native-like structure, supporting its use as a vaccine candidate.

Large-scale preparation of α-D-(14)-oligogalacturonic acids from pectic acid

2002 ◽  
Vol 80 (8) ◽  
pp. 900-903 ◽  
Author(s):  
Hong-Ni Fan ◽  
Mei-Zheng Liu ◽  
Yuan C Lee
Keyword(s):  
Galacturonic Acid ◽  
Large Scale ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Inexpensive Method ◽  
Pectic Acid ◽  
Large Scale Preparation ◽  
Exclusion Chromatography ◽  
Scale Preparation

An efficient and inexpensive method for large-scale preparation of α-D-(1[Formula: see text]4)-oligogalacturonic acids (oligo-GalA), up to DP 5, from pectic acid is described. Pectic acid was digested with a commercially available pectinase to yield a mixture of oligo-GalA, which was effectively separated by a combination of low-pressure – size-exclusion chromatography based on ion-exchange chromatography to obtain pure oligo-GalA of DP 2-5. Key words: pectic acid, galacturonic acid, galabiose, galatriose, pectinase.

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Characterization of Primary Isolate-Like Variants of Simian-Human Immunodeficiency Virus

1999 ◽  
Vol 73 (12) ◽  
pp. 10199-10207 ◽  
Author(s):  
John M. Crawford ◽  
Patricia L. Earl ◽  
Bernard Moss ◽  
Keith A. Reimann ◽  
Michael S. Wyand ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mononuclear Cells ◽  
Vaccine Development ◽  
Envelope Glycoproteins ◽  
Serum Samples ◽  
Aids Vaccine ◽  
Antigenic Properties ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Soluble Cd4

ABSTRACT Several different strains of simian-human immunodeficiency virus (SHIV) that contain the envelope glycoproteins of either T-cell-line-adapted (TCLA) strains or primary isolates of human immunodeficiency virus type 1 (HIV-1) are now available. One of the advantages of these chimeric viruses is their application to studies of HIV-1-specific neutralizing antibodies in preclinical AIDS vaccine studies in nonhuman primates. In this regard, an important consideration is the spectrum of antigenic properties exhibited by the different envelope glycoproteins used for SHIV construction. The antigenic properties of six SHIV variants were characterized here in neutralization assays with recombinant soluble CD4 (rsCD4), monoclonal antibodies, and serum samples from SHIV-infected macaques and HIV-1-infected individuals. Neutralization of SHIV variants HXBc2, KU2, 89.6, and 89.6P by autologous and heterologous sera from SHIV-infected macaques was restricted to an extent that these viruses may be considered heterologous to one another in their major neutralization determinants. Little or no variation was seen in the neutralization determinants on SHIV variants 89.6P, 89.6PD, and SHIV-KB9. Neutralization of SHIV HXBc2 by sera from HXBc2-infected macaques could be blocked with autologous V3-loop peptide; this was less true in the case of SHIV 89.6 and sera from SHIV 89.6-infected macaques. The poorly immunogenic but highly conserved epitope for monoclonal antibody IgG1b12 was a target for neutralization on SHIV variants HXBc2, KU2, and 89.6 but not on 89.6P and KB9. The 2G12 epitope was a target for neutralization on all five SHIV variants. SHIV variants KU2, 89.6, 89.6P, 89.6PD, and KB9 exhibited antigenic properties characteristic of primary isolates by being relatively insensitive to neutralization in peripheral blood mononuclear cells with serum samples from HIV-1-infected individuals and 12-fold to 38-fold less sensitive to inhibition with recombinant soluble CD4 than TCLA strains of HIV-1. The utility of nonhuman primate models in AIDS vaccine development is strengthened by the availability of SHIV variants that are heterologous in their neutralization determinants and exhibit antigenic properties shared with primary isolates.

Screening Tagged Proteins Using Tandem Affinity-Buffer Exchange Chromatography Online with Native Mass Spectrometry

2021 ◽  
Author(s):  
Florian Busch ◽  
Zachary VanAernum ◽  
Stella M. Lai ◽  
Venkat Gopalan ◽  
Vicki Wysocki
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Tertiary Structure ◽  
Direct Analysis ◽  
Native Mass Spectrometry ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Functional Protein ◽  
Translation Rate

Protein overexpression and purification are critical for in vitro structure-function characterization studies. However, some proteins are difficult to express robustly in heterologous systems due to host-related (e.g., codon usage, translation rate) and/or protein specific (e.g., toxicity, aggregation) challenges. Therefore, it is often necessary to screen<br>multiple overexpression and purification conditions to maximize the yield of functional protein, particularly for resource-heavy downstream applications (e.g., biocatalysts, tertiary structure determination, biotherapeutics). Here, we describe an automatable liquid chromatography–mass spectrometry-based method for rapid, direct analysis of target proteins in cell lysates. This online approach is facilitated by coupling immobilized metal affinity chromatography (IMAC), which leverages engineered poly-histidine tags in proteins of interest, with size exclusion-based buffer exchange (OBE) and native mass spectrometry (nMS). The use of IMAC-OBE-nMS to optimize conditions for large-scale protein production should expedite structural biology and biotherapeutic initiatives.<br>

A Combination of Cation Exchange and Ligand-Affinity Chromatography for Purification of Two Molecular Species of the Folate Binding Protein in Human Milk, One Equipped with a Hydrophobic Glycosyl Phosphatidylinositol Tail: Characterization of Hydrophobicity and Electrical Charge

2002 ◽  
Vol 22 (3-4) ◽  
pp. 443-454 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Human Milk ◽  
Affinity Chromatography ◽  
Cation Exchange ◽  
Large Scale ◽  
Gel Filtration ◽  
Molecular Size ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Affinity Column ◽  
Folate Binding Protein

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5–6 was a cleavage product of a 100 kDa protein eluted at pH 3–4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7–9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

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