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1.
Mol Cell ; 57(5): 769-783, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25620564

RESUMEN

Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.


Asunto(s)
Diferenciación Celular , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Células HEK293 , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Ratones Noqueados , Modelos Genéticos , Mutación , Complejo Represivo Polycomb 2/genética , Interferencia de ARN
2.
PLoS Biol ; 11(9): e1001666, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24086110

RESUMEN

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Metabolismo Energético/genética , Metabolismo de los Lípidos/genética , Miocardio/metabolismo , Consumo de Oxígeno/genética , Adenosina Trifosfato/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Hipoxia de la Célula/genética , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Corazón/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Oxígeno/metabolismo , Activación Transcripcional
3.
J Pept Sci ; 22(1): 4-27, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26785684

RESUMEN

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very-high-quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications.


Asunto(s)
Aminoácidos/química , Fluorenos/química , Péptidos/síntesis química , Procesamiento Proteico-Postraduccional , Técnicas de Síntesis en Fase Sólida/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Línea Celular , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glicosilación , Humanos , Metilación , Péptidos/farmacología , Fosforilación , Prenilación de Proteína
4.
J Pept Sci ; 22(5): 360-7, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-27086749

RESUMEN

A backbone amide bond protecting group, 2-hydroxy-4-methoxy-5-nitrobenzyl (Hmnb), improved the synthesis of aggregation and aspartimide-prone peptides. Introduction of Hmnb is automated and carried out during peptide assembly by addition of 4-methoxy-5-nitrosalicylaldehyde to the peptidyl-resin and on-resin reduction to the secondary amine. Acylation of the hindered secondary amine is aided by the formation of an internal nitrophenol ester that undergoes a favourable O,N intramolecular acyl transfer. This activated ester participates in the coupling and generally gives complete reaction with standard coupling conditions. Hmnb is easily available in a single preparative step from commercially available material. Different methods for removing the amide protecting group were explored. The protecting group is labile to acidolysis, following reduction of the nitro group to the aniline. The two main uses of backbone protection of preventing aspartimide formation and of overcoming difficult sequences are demonstrated, first with the synthesis of a challenging aspartimide-prone test sequence and then with the classic difficult sequence ACP (65-74) and a 23-mer homopolymer of polyalanine.


Asunto(s)
Amidas/química , Ácido Aspártico/análogos & derivados , Péptidos/síntesis química , Acilación , Secuencia de Aminoácidos , Ácido Aspártico/química , Estructura Molecular , Nitrobencenos/química , Péptidos/química , Técnicas de Síntesis en Fase Sólida
5.
Angew Chem Int Ed Engl ; 55(42): 13174-13179, 2016 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-27654901

RESUMEN

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post-translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.


Asunto(s)
Ésteres/química , Ésteres del Ácido Fórmico/síntesis química , Péptidos/química , Compuestos de Sulfhidrilo/química , Ciclización , Ésteres del Ácido Fórmico/química , Estructura Molecular
6.
Nature ; 446(7139): 1038-45, 2007 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-17460665

RESUMEN

The sustained effort towards developing an antibody vaccine against HIV/AIDS has provided much of our understanding of viral immunology. It is generally accepted that one of the main barriers to antibody neutralization of HIV is the array of protective structural carbohydrates that covers the antigens on the virus's surface. Intriguingly, however, recent findings suggest that these carbohydrates, which have evolved to protect HIV and promote its transmission, are also attractive therapeutic targets.


Asunto(s)
Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Carbohidratos/inmunología , Diseño de Fármacos , VIH-1/química , VIH-1/inmunología , Animales , Fármacos Anti-VIH/farmacología , Carbohidratos/biosíntesis , Carbohidratos/química , Antígenos VIH/química , Antígenos VIH/inmunología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos
7.
Nature ; 448(7149): 87-91, 2007 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-17589501

RESUMEN

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.


Asunto(s)
Proteínas de Unión al ADN/química , Histonas/metabolismo , Oxidorreductasas N-Desmetilantes/química , Factores de Transcripción/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas con Dominio de Jumonji , Modelos Moleculares , Oxidorreductasas N-Desmetilantes/metabolismo , Conformación Proteica , Proteínas Recombinantes , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Especificidad por Sustrato , Factores de Transcripción/metabolismo
8.
Front Sociol ; 8: 1076750, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37139226

RESUMEN

On occasion it makes sound sense to undertake a retrospective review of a late colleague's contribution to his or her subject area. This applies to Robert Pinker, Professor of Social Administration at the London School of Economics, who died at the age of 89 in February 2021. Over a long life he made a major impact on working for press freedom and to social work studies, but this article concerns his work on social policy, and particularly on the idea of welfare pluralism, a many-faceted idea the exploration of which powered two pathbreaking books Social Theory and Social Policy (1971) and The Idea of Welfare (1979). In the twentieth century many states including the United Kingdom had greatly expanded their welfare provisions for their citizens, and, in some, an academic subject area, often called social administration or social policy had grown in response. Pinker started writing in the 1960s, dissatisfied with the conventional approach of Richard Titmuss and others, almost exclusively concerned with the state and welfare. He made the case for a radical rebalance toward including everyday experiences of obligations and how familial informal welfare practices are strengthened, weakened or modified by formal social services. However, ahead of his time, Pinker was arguing for an enhanced sociological imagination in the study of social policy and on the very idea of "welfare". This article has sections reflecting the facets of Pinker's thinking about welfare pluralism, including "social policy's past", "exchange and stigma", "taking informal welfare seriously", "divergent views of altruism", "comparative studies", "on a mixture of means to welfare" and "aspects of Pinker's legacy". The idea of welfare pluralism is now familiar. But Pinker's crucial pioneering role, depth of understanding of the issues and grasp of their intertwining is seldom recalled. This article should help to meet the need for his contribution to be reinserted into the mainstream of sociological thought on welfare, so enriching new research.

9.
Mol Membr Biol ; 28(5): 254-64, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21604991

RESUMEN

Abstract The hepatitis C virus (HCV) encodes the p7 protein that oligomerizes to form an ion channel. The 63 amino acid long p7 monomer is an integral membrane protein predominantly found in the endoplasmic reticulum (ER). Although it is currently unknown whether p7 is incorporated into secreted virions, its presence is crucial for the release of infectious virus. The molecular and biophysical mechanism employed by the p7 ion channel is largely unknown, but in vivo it is likely to be embedded in membranes undergoing changes in lipid composition. In this study we analyze the influence of the lipid environment on p7 ion channel structure and function using electrophysiology and synchrotron radiation circular dichroism (SRCD) spectroscopy. We incorporated chemically synthesized p7 polypeptides into artificial planar membranes of various lipid compositions. A lipid bilayer composition comprising phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (4:1 PC:PE) led to burst-like patterns in the channel recordings with channel openings lasting up to 0.5 s. The reverse ratio of PC:PE (1:4) gave rise to individual channels continuously opening for up to 8 s. SRCD spectroscopy of p7 embedded into liposomes of corresponding lipid compositions suggests there is a structural effect of the lipid composition on the p7 protein.


Asunto(s)
Hepacivirus/metabolismo , Canales Iónicos/química , Canales Iónicos/metabolismo , Lípidos/química , Proteínas Virales/química , Proteínas Virales/metabolismo , Dicroismo Circular , Activación del Canal Iónico , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Relación Estructura-Actividad , Sincrotrones
10.
Methods Mol Biol ; 2208: 1-12, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32856252

RESUMEN

Peptide thioesters serve as fundamental building blocks for the synthesis of proteins and cyclic peptides. Classically, methods to synthesize thioesters have been based on acid-labile amino-protecting groups for which final side-chain deprotection required the use of hazardous hydrogen fluoride (HF). Alternative protection schemes based on base-labile amino-protecting groups have become preferred methods but are not suitable due to the lability of thioester bonds toward bases. In this method, we employ a trifluoracetic acid/trimethylsilyl bromide (TFA/TMSBr) protocol using a hydroxymethyl resin obviating the need for HF. TFA/TMSBr is volatile enough to be easily removed yet less hazardous than HF, making it more practical for general peptide chemists. We describe optimized cleavage procedures and appropriate protecting group schemes and discuss in situ neutralization protocols. The method is relatively simple, straightforward, and easily scalable, allowing the facile preparation of alkyl and aryl thioesters.


Asunto(s)
Ésteres/química , Péptidos/química , Compuestos de Azufre/química , Ácido Fluorhídrico/química , Compuestos de Trimetilsililo/química
11.
Methods Mol Biol ; 2103: 225-237, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31879929

RESUMEN

Solubility is a key property of peptides and of central importance to the success of solid-phase peptide synthesis and subsequent peptide purification and handling. Substitution of the backbone amide bond can dramatically increase peptide solubility. Backbone amide bond protection works by preventing the formation of interchain association and can be used both to synthesize aggregation-prone peptide sequences on solid phase and to improve solubility of a peptide post synthesis. Improving peptide solubility by judicial use of backbone protection is of growing importance, particularly for chemical protein synthesis by chemical ligation.


Asunto(s)
Amidas , Aminoácidos/química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Amidas/química , Secuencia de Aminoácidos , Benzaldehídos/química , Cromatografía Líquida de Alta Presión , Microondas , Péptidos/química , Péptidos/aislamiento & purificación , Técnicas de Síntesis en Fase Sólida/instrumentación , Solubilidad
12.
Chembiochem ; 10(8): 1340-3, 2009 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-19402090

RESUMEN

Less than 6 feet under: Serum proteins C3, C4, and alpha(2)M each contain a thioester domain buried within a hydrophobic pocket, which is thought to shield the labile thioester from hydrolysis. Herein, we make use of the inherent reactivity of the hydrazide for thioester moieties to chemoselectively label these crucial serum regulators in their native conformation; this demonstrates that access to the thioester site is much greater than previously supposed.


Asunto(s)
Complemento C3/química , Complemento C4b/química , Compuestos de Sulfhidrilo/química , alfa-Macroglobulinas/química , Biotina/química , Complemento C3/inmunología , Complemento C4b/inmunología , Colorantes Fluorescentes/química , Péptidos/química , Ingeniería de Proteínas , alfa-Macroglobulinas/inmunología
13.
Front Sociol ; 4: 1, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-33869330

RESUMEN

This article attempts to retrieve important aspects of Spencer's sociology from the general neglect and misrepresentation which threatens to overwhelm it all. It does touch in passing on many such highly dubious contentions as that he was a "social Darwinist," but the prime focus is to deal with three linked themes. First, the article examines the significance of his attribution to individuals of "social self-consciousness" as part of sociality, thus distancing it from Durkheim's influential but suspect reading of Spencer's individuals as egoistic. Second, it rescues his concept of "the social organism" from misinterpretation. His own writings show it to be a more rigorous and suggestive attempt to configure the morphology of "the social" than commonly assumed. Third, it reconstructs the status of his contrast between "militant" and "industrial" social forms as a contrast between different but more general forms of social life that those descriptions in fact register. With the focus on these three linked themes the article improves the historical accuracy of our understanding of Spencer's sociology. It also repositions key aspects of it as not alien, quaint and a spent force, but ontologically challenging and possibly prescient for debates about the meaning of "the social" today.

14.
Front Sociol ; 4: 77, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-33869399

RESUMEN

This article presents new insights into Spencer's theoretical sociology as he applied it to the professions and professional institutions, which he discussed extensively, particularly in his Principles of Sociology. The first part of this article notes the main conceptual insights which he established and aligns them within the wider context of a re-reading of Spencer's sociology. Particular attention is paid to the "social organism" and the spontaneous cooperation of social individuals in society (with each possessing "social self-consciousness"). This part also reappraises Spencer's account of the emergence of "professionals" and their distinctive "cunning, skill, and acquaintance with the nature of things," which professionals have brought to bear on what has been experienced in the ordinary social lives of people as complexity or the unfamiliar in the world. The subsequent discussion focuses on, first, a retrieval of Spencer's theoretical stance on the activities of the professions, and on work and conditions in general, and, second, on reviewing some of the major resonances which his work has with practical problems and the associated theoretical issues concerning the sociological understanding of professional/service-user interaction in social life today.

15.
Chem Commun (Camb) ; 55(31): 4566-4569, 2019 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-30931466

RESUMEN

A series of cyclic lipidated oligo-Arg cell penetrating peptides were synthesised with varied macrocycle size and lipid chain anchoring site. The study of their cellular uptake revealed different structural requirements to promote efficient glycosaminoglycan-dependent endocytosis and direct translocation.


Asunto(s)
Péptidos de Penetración Celular/química , Glicosaminoglicanos/química , Secuencia de Aminoácidos , Animales , Células CHO , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/metabolismo , Cricetinae , Cricetulus , Ciclización , Endocitosis
16.
Commun Chem ; 2: 111, 2019 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-31942456

RESUMEN

The ubiquitylation of NF-κB essential modulator (NEMO) is part of the intracellular immune signalling pathway. Monoubiquitylated NEMO is required for exploring the mechanism of NEMO linear ubiquitylation by LUBAC (linear ubiquitin chain assembly complex), but is not accessible by biological techniques. Here we perform the chemical ubiquitylation of NEMO using a ligation auxiliary, which only requires a two-step synthesis, and is easily installed onto the lysine side-chain. Chemical ligation occurs directly on the lysine ε amine and remains efficient below pH 7. We show that ubiquitylated NEMO has similar affinity to linear diubiquitin chains as unmodified NEMO. The proximal ubiquitin of chemically synthesised NEMOCoZi-Ub is accepted as a substrate for linear extension by the (RING-Between-RING) RBR domain of HOIL-1-interacting protein (HOIP) alone. Our results indicate that NEMO linear ubiquitylation consists of two-steps, an initial priming event and a separate extension step requiring different LUBAC components.

18.
Sci Rep ; 7(1): 14083, 2017 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-29074846

RESUMEN

Most eukaryotic RNA regulators recognise their RNA and protein partners by the combinatorial use of several RNA binding domains. Inter-domain dynamics and interactions play a key role in recognition and can be analysed by techniques such as NMR or FRET, provided that the information relative to the individual interactions can be de-convoluted. Segmentally labelling the proteins by ligating labelled and unlabelled peptide chains allows one to filter out unwanted information and observe the labelled moieties only. Several strategies have been implemented to ligate two protein fragments, but multiple ligations, which are necessary to segmentally label proteins of more than two domains, are more challenging and often dependent on the structure and solubility of the domains. Here we report a method to ligate multiple protein segments that allows the fast, high yield labelling of both internal and end domains, depending on the requirements. We use TCEP and mercaptophenylacetic acid (MPAA) in an optimised reaction environment to achieve an efficient ligation of protein domains independently from their structure or solubility. We expect the method will provide a useful tool for the molecular study of combinatorial protein-RNA recognition in RNA regulation.


Asunto(s)
Técnicas de Sonda Molecular , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/aislamiento & purificación , Escherichia coli , Humanos , Espectrometría de Masas , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína SUMO-1/química , Proteína SUMO-1/metabolismo
19.
J Immunol Methods ; 281(1-2): 143-8, 2003 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-14580888

RESUMEN

Immobilized metal affinity chromatography (IMAC) has rapidly become one of the most widespread affinity purification techniques employed in recombinant protein expression. However, the high purity demands of certain applications are occasionally unattainable through a single IMAC separation. GNC92H2scFv is a cocaine-binding single-chain antibody fragment that is unstable during long-term storage in aqueous solution. To circumvent this problem, a reversed-phase HPLC separation was performed following IMAC purification of GNC92H2scFv from Escherichia coli cell culture supernatant. The resulting HPLC effluent was then freeze-dried to afford a salt-free lyophilizate amenable to long-term storage with minimal loss in binding activity. HPLC purification also effectively removed an 80-kDa protein contaminant that co-eluted with the IMAC-purified protein. Of special importance for in vivo applications of recombinantly expressed protein therapeutics, an HPLC purification step afforded a 1000-fold reduction in lipopolysaccharide (LPS) endotoxin contamination in the final GNC92H2scFv product.


Asunto(s)
Cocaína/metabolismo , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Fragmentos de Inmunoglobulinas/metabolismo , Lipopolisacáridos/aislamiento & purificación
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