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1.
J Biol Chem ; 288(5): 3251-64, 2013 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-23250752

RESUMEN

Two-pore domain potassium (K(2P)) channels play fundamental roles in cellular processes by enabling a constitutive leak of potassium from cells in which they are expressed, thus influencing cellular membrane potential and activity. Hence, regulation of these channels is of critical importance to cellular function. A key regulatory mechanism of K(2P) channels is the control of their cell surface expression. Membrane protein delivery to and retrieval from the cell surface is controlled by their passage through the secretory and endocytic pathways, and post-translational modifications regulate their progression through these pathways. All but one of the K(2P) channels possess consensus N-linked glycosylation sites, and here we demonstrate that the conserved putative N-glycosylation site in K(2P)3.1 and K(2P)9.1 is a glycan acceptor site. Patch clamp analysis revealed that disruption of channel glycosylation reduced K(2P)3.1 current, and flow cytometry was instrumental in attributing this to a decreased number of channels on the cell surface. Similar findings were observed when cells were cultured in reduced glucose concentrations. Disruption of N-linked glycosylation has less of an effect on K(2P)9.1, with a small reduction in number of channels on the surface observed, but no functional implications detected. Because nonglycosylated channels appear to pass through the secretory pathway in a manner comparable with glycosylated channels, the evidence presented here suggests that the decreased number of nonglycosylated K(2P)3.1 channels on the cell surface may be due to their decreased stability.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Glucosa/farmacología , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación/efectos de los fármacos , Células HEK293 , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso , Canales de Potasio de Dominio Poro en Tándem/química , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Alineación de Secuencia , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo
2.
J Biol Chem ; 286(16): 14110-9, 2011 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-21357689

RESUMEN

Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background "leak" channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K(+) flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by ßCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas del Tejido Nervioso/química , Canales de Potasio de Dominio Poro en Tándem/química , Animales , Membrana Celular/metabolismo , Chaperoninas/química , Electrofisiología , Proteínas Fluorescentes Verdes/química , Humanos , Fosforilación , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina/química , Xenopus laevis
3.
Immunology ; 136(2): 163-75, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22260486

RESUMEN

Cross-presentation is the presentation by MHC class I of antigenic peptides from exogenous proteins that have been internalized and processed by professional antigen-presenting cells, e.g. dendritic cells. We have investigated the influence of particle size and antigen load on cross-presentation following antigen delivery on microspheres (MS). Cross-presentation from small particles (0·8-µm) is sensitive to proteasome inhibition and the blockade of endoplasmic reticulum-resident MHC class I complex export, whereas cross-presentation from larger particles (aggregated clumps of 0·8-µm MS) is resistant to these antagonists. This observation may have been overlooked previously, because of the heterogeneity of particle size and MS uptake in unsorted dendritic cell populations. Larger particles carry more antigen, but we show that antigen load does not influence the cross-presentation pathway used. Whereas early endosome autoantigen 1 (EEA1) could be observed in all phagosomes, we observed endoplasmic reticulum SNARE of molecular weight 24 000 (ERS24) and cathepsin S in association with 3·0-µm and aggregated 0·8-µm MS, but not individual 0·8-µm MS. A potential mechanism underlying our observations may be the activation of ß-catenin by disruption of E-cadherin-mediated adhesion. Activated ß-catenin was detected in the cytoplasm of cells after phagocytosis of MS (highest levels for the largest particles). We propose that particle size can direct the use of different pathways for the cross-presentation of an identical antigen. Furthermore, these pathways have differing yields of MHC class I-peptide complexes, which is an important variable in designing vaccination strategies for maximal antigen expression and CD8(+) T-cell priming.


Asunto(s)
Reactividad Cruzada/inmunología , Tamaño de la Partícula , Fagocitosis/inmunología , Animales , Catepsinas/inmunología , Línea Celular , Ratones , Microesferas , Fagosomas/inmunología , Proteínas R-SNARE/inmunología , Proteínas de Transporte Vesicular/inmunología , beta Catenina/inmunología
5.
Front Med Technol ; 3: 664259, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35047921

RESUMEN

Introduction: SARS-CoV-2 infection is a global pandemic. Personal Protective Equipment (PPE) to protect healthcare workers has been a recurrent challenge in terms of global stocks, supply logistics and suitability. In some settings, around 20% of healthcare workers treating COVID-19 cases have become infected, which leads to staff absence at peaks of the pandemic, and in some cases mortality. Methods: To address shortcomings in PPE, we developed a simple powered air purifying respirator, made from inexpensive and widely available components. The prototype was designed to minimize manufacturing complexity so that derivative versions could be developed in low resource settings with minor modification. Results: The "Personal Respirator - Southampton" (PeRSo) delivers High-Efficiency Particulate Air (HEPA) filtered air from a battery powered fan-filter assembly into a lightweight hood with a clear visor that can be comfortably worn for several hours. Validation testing demonstrates that the prototype removes microbes, avoids excessive CO2 build-up in normal use, and passes fit test protocols widely used to evaluate standard N95/FFP2 and N99/FFP3 face masks. Feedback from doctors and nurses indicate the PeRSo prototype was preferred to standard FFP2 and FFP3 masks, being more comfortable and reducing the time and risk of recurrently changing PPE. Patients report better communication and reassurance as the entire face is visible. Conclusion: Rapid upscale of production of cheaply produced powered air purifying respirators, designed to achieve regulatory approval in the country of production, could protect healthcare workers from infection and improve healthcare delivery during the COVID-19 pandemic.

6.
Biomaterials ; 27(30): 5299-306, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16808972

RESUMEN

A polymer microarray of 120 polyurethanes was used to identify polymers that promoted the adhesion of bone marrow dendritic cells (BMDC). Identified polymers were coated onto glass cover slips and shown to be efficient substrates for the immobilisation of these primary cells, which underwent efficient phagocytosis while still presumably maintaining their immature state.


Asunto(s)
Bioensayo , Células de la Médula Ósea , Células Dendríticas , Fagocitosis , Poliuretanos/química , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Adhesión Celular , Células Inmovilizadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Poliuretanos/síntesis química , Poliuretanos/farmacología
7.
FEBS J ; 272(15): 4002-10, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16045770

RESUMEN

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Tilacoides/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Péptido Hidrolasas , Complejo de Proteína del Fotosistema I/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coloración y Etiquetado
8.
FEBS Lett ; 565(1-3): 101-5, 2004 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-15135060

RESUMEN

An Arabidopsis cDNA coding for a previously uncharacterized isoform of inorganic pyrophosphatase was isolated. It was used to complement an E. coli mutant, demonstrating that it coded for an active enzyme. MgCl(2) was necessary for the protein's activity, whilst NaF inhibited it. The K(m) for pyrophosphate and the pH optimum of the protein was determined. The gene coding for this protein was expressed in all tissues, and its expression in rosette leaves was induced by incubation on metabolizable sugars. In vitro import experiments demonstrated that the protein could be imported into chloroplasts and localized to the stromal compartment.


Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Cloroplastos/enzimología , Pirofosfatasa Inorgánica/biosíntesis , Pirofosfatasa Inorgánica/química , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Cinética , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Pisum sativum/enzimología , Filogenia , Plastidios/metabolismo , Isoformas de Proteínas , Transporte de Proteínas , ARN/química , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Fluoruro de Sodio/farmacología , Distribución Tisular
9.
FEBS Lett ; 510(3): 145-8, 2002 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-11801243

RESUMEN

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.


Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Subunidades de Proteína , Secuencia de Aminoácidos , Arabidopsis , Cloroplastos/química , Cloroplastos/metabolismo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Hordeum , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Peso Molecular , Complejo de Proteína del Fotosistema I , Proteínas de Plantas/química , Proteínas de Plantas/genética , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de Proteína , Spinacia oleracea
10.
J Histochem Cytochem ; 52(5): 701-4, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15100247

RESUMEN

We developed a rapid and reliable technique for specifically staining intact chloroplasts using the fluorescent dye carboxyfluorescein diacetate. Intact, import-competent chloroplasts were isolated simply and rapidly from soil-grown Arabidopsis thaliana plants, with yields of 20 +/- 5 micro g chlorophyll per g FW, greater than previously reported yields from soil-grown Arabidopsis. Traditional chloroplast isolation buffers sometimes contain low concentrations (<10 mM) sodium ascorbate as a general-purpose anti-oxidant, but we found that only Arabidopsis chloroplasts isolated in the presence of high concentrations (50-100 mM) of sodium ascorbate in the initial grinding buffer were import-competent.


Asunto(s)
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Colorantes Fluorescentes/metabolismo , Orgánulos/metabolismo , Arabidopsis/ultraestructura , Ácido Ascórbico , Tampones (Química) , Separación Celular , Fluoresceínas/metabolismo , Microscopía Fluorescente , Pisum sativum , Suelo
11.
Channels (Austin) ; 7(4): 288-302, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23807092

RESUMEN

Acid-sensitive, two-pore domain potassium channels, K(2P)3.1 and K(2P)9.1, are implicated in cardiac and nervous tissue responses to hormones, neurotransmitters and drugs. K(2P)3.1 and K(2P)9.1 leak potassium from the cell at rest and directly impact membrane potential. Hence altering channel number on the cell surface drives changes in cellular electrical properties. The rate of K(2P)3.1 and K(2P)9.1 delivery to and recovery from the plasma membrane determines both channel number at the cell surface and potassium leak from cells. This study examines the endocytosis of K(2P)3.1 and K(2P)9.1. Plasma membrane biotinylation was used to follow the fate of internalized GFP-tagged rat K(2P)3.1 and K(2P)9.1 transiently expressed in HeLa cells. Confocal fluorescence images were analyzed using Imaris software, which revealed that both channels are endocytosed by a dynamin-dependent mechanism and over the course of 60 min, move progressively toward the nucleus. Endogenous endocytosis of human K(2P)3.1 and K(2P)9.1 was examined in the lung carcinoma cell line, A549. Endogenous channels are endocytosed over a similar time-scale to the channels expressed transiently in HeLa cells. These findings both validate the use of recombinant systems and identify an endogenous model system in which K(2P)3.1 and K(2P)9.1 trafficking can be further studied.


Asunto(s)
Dinaminas/metabolismo , Endocitosis , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Endosomas/metabolismo , Células HeLa , Humanos , Cinética , Proteínas del Tejido Nervioso , Ratas
12.
J Biol Chem ; 281(15): 10548-54, 2006 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-16478728

RESUMEN

The PSI-G subunit of photosystem I (PSI) is an 11-kDa membrane protein that plays an important role in electron transport between plastocyanin and PSI and is involved in the stability of the PSI complex. Within the complex, the PSI-G subunit is bound to PSI-B and is in contact with Lhca1. PSI-G has two transmembrane spans connected by a positively charged stromal loop. The loop is inaccessible to proteases, indicating a tightly bound location within the PSI complex. Here, we have studied the insertion mechanism and assembly of PSI-G. We show that the protein inserts into thylakoids by a direct or "spontaneous" pathway that does not involve the activities of any known chloroplast protein-targeting machinery. Surprisingly, the positively charged stromal loop region plays a major role in this process. Mutagenesis or deletions within this region almost invariably lead to a marked lowering of insertion efficiency, strongly indicating a critical role for the loop in the organization of the transmembrane regions prior to or during membrane insertion. Finally, we have examined the assembly of newly inserted PSI-G into the PSI complex, since very little is known of the assembly pathway for this large multimeric complex. Interestingly, we find that inserted PSI-G can be found within the full PSI complex within the import assay time frame after insertion into thylakoids, strongly suggesting that PSI-G normally associates at the end of the assembly process. This is consistent with its location on the periphery of the complex.


Asunto(s)
Complejo de Proteína del Fotosistema I/fisiología , Tilacoides/metabolismo , Adenosina Trifosfato/química , Membrana Celular/metabolismo , Cloroplastos/metabolismo , ADN/química , Transporte de Electrón , Electroforesis en Gel Bidimensional , Eliminación de Gen , Concentración de Iones de Hidrógeno , Modelos Biológicos , Mutagénesis , Nucleótidos/química , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejo de Proteína del Fotosistema I/química , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Protones , Tilacoides/enzimología
13.
Proc Natl Acad Sci U S A ; 102(5): 1785-90, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15665090

RESUMEN

The recently discovered potato tuber (Solanum tuberosum) alpha-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 microM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.


Asunto(s)
Fosfotransferasas (Aceptores Pareados)/química , Fosfotransferasas (Aceptores Pareados)/metabolismo , Plastidios/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Oscuridad , Disulfuros/metabolismo , Ditiotreitol/farmacología , Activación Enzimática/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Hojas de la Planta/enzimología , Unión Proteica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spinacia oleracea/enzimología , Almidón/biosíntesis
14.
Plant Cell ; 15(1): 133-49, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12509527

RESUMEN

Isoamylases are debranching enzymes that hydrolyze alpha-1,6 linkages in alpha-1,4/alpha-1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.


Asunto(s)
Glucanos/metabolismo , Isoamilasa/genética , Isoamilasa/metabolismo , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Catálisis , Activación Enzimática/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tallos de la Planta/enzimología , Tallos de la Planta/genética , Homología de Secuencia de Aminoácido , Solanum tuberosum/genética , Almidón/biosíntesis
15.
J Cell Sci ; 117(Pt 20): 4807-18, 2004 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-15340011

RESUMEN

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Cloroplastos/fisiología , Dedos de Zinc , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Prueba de Complementación Genética , Péptidos y Proteínas de Señalización Intracelular , Luz , Datos de Secuencia Molecular , Fenotipo , Pigmentación , Pigmentos Biológicos/metabolismo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos Híbridos
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