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No abstract
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Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk, et al., 1992, Biochem. J. 286, 793-800; B. Fan et al., 1993, J. Biol. Chem. 268, 17588-17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.
Asunto(s)
Antitrombina III/metabolismo , Heparina/metabolismo , Animales , Antitrombina III/química , Asparagina/química , Sitios de Unión , Línea Celular , Cricetinae , Glutamina/química , Glicosilación , Humanos , Cinética , Mesocricetus , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We have analysed the status and transcriptional activity of the bovine papillomavirus-I (complete BPV-I genome)-based expression vector pCES in CI27i-cell-line-derived 3TI cells used for the industrial production of recombinant human erythropoietin (rhuEpo). Complete tandem head-to-tail integration of about 600 vector copies at a single site of the cellular genome was observed. Deletions, insertions or rearrangements of pCES-specific sequences or extrachromosomal copies of vector sequences were not detected. Transcriptional analyses demonstrated that rhuEpo was abundantly expressed. BPV-I early transcription was detected, as expected from a BPV-I-transformed cell line; however, compared with the mouse metallothionein-I-promoter-driven rhuEpo-specific transcription, it was very weak. Late BPV-I transcription was also not detected in 3TI cells under conditions of large-scale rhuEpo production. Therefore this expression system proved to be safe and suitable for the production of rhuEpo.
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Papillomavirus Bovino 1/genética , Eritropoyetina/genética , Vectores Genéticos , Genoma Viral , Transcripción Genética , Animales , Secuencia de Bases , Células Clonales , Eritropoyetina/biosíntesis , Fermentación , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Células Tumorales CultivadasRESUMEN
The human beta-trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK-21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT-beta TP or pAB3-1 which contain the cDNAs encoding the human secretory glycoproteins beta-trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP-NeuAc:Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase (ST6N) gene. The beta-trace protein was purified by immunoaffinity chromatography and N-linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural beta-trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of beta-trace protein coexpressed with human ST6N were found to contain NeuAc in alpha 2,6- or alpha 2,3-linkage in the same ratio. From 1H-NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both alpha 2,3- and alpha 2,6-linkage to Gal beta(1-4)GlcNAc-R, whereas 40% of the molecules of this fraction contained NeuAc in only alpha 2,3-linkage to Gal(beta 1-4)GlcNAc-R. The alpha 2,6-linked NeuAc was shown to be attached preferentially to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human-type glycosylated secretory recombinant polypeptides.
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Glicoproteínas/biosíntesis , Oxidorreductasas Intramoleculares , Proteínas Recombinantes/biosíntesis , Sialiltransferasas/metabolismo , Animales , Antitrombina III/biosíntesis , Secuencia de Bases , beta-Globulinas/biosíntesis , beta-Globulinas/química , beta-Globulinas/metabolismo , Secuencia de Carbohidratos , Línea Celular , Clonación Molecular , Cricetinae , Glicoproteínas/química , Glicoproteínas/metabolismo , Humanos , Lipocalinas , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferasas/química , Sialiltransferasas/genética , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.
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Eritropoyetina/análogos & derivados , Eritropoyetina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Secuencia de Bases , Unión Competitiva , Conformación de Carbohidratos , Línea Celular , Cricetinae , Glicosilación , Humanos , Riñón , Mesocricetus , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Receptores de Eritropoyetina/metabolismo , Tasa de Secreción , Relación Estructura-ActividadRESUMEN
To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin (rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced almost identical NMR perturbation difference spectra. The only functional difference found was in the rates of inactivation of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I gave a biphasic inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin affinity and can alter the rate of proteinase inhibition.
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Antitrombina III/metabolismo , Heparina/metabolismo , Animales , Antitrombina III/química , Carbohidratos/análisis , Línea Celular , Cricetinae , Electroforesis en Gel de Poliacrilamida , Inhibidores del Factor Xa , Glicosilación , Heparina/química , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.
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Antivirales/metabolismo , Antígenos CD4/metabolismo , VIH-1/efectos de los fármacos , Inmunoglobulina G/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Animales , Antivirales/aislamiento & purificación , Antivirales/farmacología , Peso Corporal , Antígenos CD4/genética , Antígenos CD4/farmacología , Línea Celular , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/ultraestructura , VIH-1/inmunología , VIH-1/ultraestructura , Semivida , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Macaca mulatta , Tasa de Depuración Metabólica , Microscopía Inmunoelectrónica , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Subgrupos de Linfocitos T/efectos de los fármacos , Factores de Tiempo , Virión/efectos de los fármacos , Virión/inmunología , Virión/ultraestructuraRESUMEN
Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd approximately 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd approximately 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proportion of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.
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Antitrombinas/metabolismo , Heparina/metabolismo , Animales , Células CHO , Células Cultivadas , Cromatografía de Afinidad , Cricetinae , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Glicosilación , Humanos , Cinética , Oligosacáridos/análisis , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
We studied 684 sera obtained from 20 hemophilia patients with AIDS/AIDS-related complex (ARC), 89 asymptomatic HIV+, 76 HIV- hemophilia patients and 151 healthy controls for antibodies against recombinant CD4 (rCD4). Twenty-two percent of AIDS/ARC patients, 10% of asymptomatic HIV+ patients, 17% of HIV-patients, and 1% of healthy controls had anti-rCD4 antibodies. Purified anti-rCD4 antibodies did not react with human CD4+ lymphocytes. This may explain why formation of anti-rCD4 antibodies correlated neither with the occurrence of autoantibodies against CD4+ lymphocytes nor with a decrease in CD4+ cell counts. Antibodies that were eluted from CD4+ lymphocytes after sequential adsorption and elution with separated CD8+ and CD4+ cells reacted with CD4+ lymphocytes of only some healthy individuals, suggesting diversity of CD4 expression.
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Autoanticuerpos/sangre , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/transmisión , Hemofilia A/inmunología , Reacción a la Transfusión , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adsorción , Reacciones Antígeno-Anticuerpo/inmunología , Epítopos/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/complicaciones , Hemofilia A/complicaciones , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptores del VIH/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taq-polymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121 degrees C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10(3) per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120 degrees C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes.
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ADN Recombinante/metabolismo , Eritropoyetina/genética , Plásmidos , Animales , Secuencia de Bases , División Celular , Línea Celular , Eritropoyetina/biosíntesis , Escherichia coli/genética , Fermentación , Amplificación de Genes , Vectores Genéticos , Humanos , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/biosíntesis , Mapeo RestrictivoRESUMEN
Five different peptides (P1: 84-95; P2: 152-166; P3: 52-63; P4: 7-23; P5: 110-123) homologous to relatively hydrophilic regions of human erythropoietin (huEpo) have been synthesized to identify biologically active domains of the hormone. All peptides were able to induce high titers of peptide-specific antibodies in rabbits. Antisera from rabbits induced by recombinant huEpo (rhuEpo) contained a relatively high amount of antibodies preferentially directed against three peptides (P2, P4, and P5), of which P4 comprised the amino-terminal region, P2 the carboxyl-terminus, and P5 an interior region previously described as the receptor-binding site. The same three peptides were able to induce rhuEpo-specific antibodies, whereas P1 and P3 lacked this activity. Only peptide-P2-induced antisera inhibited the biologic activity of rhuEpo in a cell proliferation assay, indicating that the carboxyl-terminal region of the molecule is essentially involved in the biologic function of rhuEpo.
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Receptores de Superficie Celular/ultraestructura , Secuencia de Aminoácidos , Aminoácidos/análisis , Procesamiento Automatizado de Datos , Electroforesis/métodos , Epítopos/genética , Eritropoyetina/metabolismo , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de Eritropoyetina , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Normal hemostasis in humans requires the interaction of a large number of plasma glycoproteins with blood platelets and vascular endothelial cells. Many of the plasma glycoproteins which participate in blood coagulation are zymogens of enzymes that interact in a stepwise manner in a series of reactions. In the last years most of these glycoproteins have been purified from human plasma by standard techniques. Some of them are used as therapeutics for restoring coagulation disorders. The knowledge about the plasma proteins involved in blood coagulation was greatly increased after cloning and sequencing of the respective complementary DNAs. Furthermore, recombinant DNA technology is used for the alternative production of several coagulation factors. It is the aim of this article to give an overview about the molecular biology of the enzymes and cofactors involved in blood coagulation.
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Factores de Coagulación Sanguínea/genética , Coagulación Sanguínea/genética , Coagulación Sanguínea/fisiología , Proteínas Sanguíneas/genética , HumanosRESUMEN
Different chimeric antibody-like molecules consisting of the four human CD4 extracellular domains (amino acids 1-369) fused to different parts of human IgG1 and IgM heavy-chain constant regions have been created and expressed in mammalian cells. For both IgG1 and IgM fusion proteins, the best expression in COS cells was observed for molecules lacking the CH1 domain of the heavy-chain constant region. The chimeric molecules are potent inhibitors of human immunodeficiency virus (HIV) infection and HIV-mediated cytotoxicity. A CD4:IgG1 hinge fusion protein, which was analyzed in more detail, binds efficiently to HIV gp160 and human Fc receptors and shows complement-assisted inhibition of viral propagation in culture. Half-life studies after intravenous application of the latter human fusion protein into mice and monkeys showed significant prolongation of serum survival compared to soluble CD4. An IgG2b murine homolog of the human CD4:IgG1 hinge fusion protein was prepared and evaluated in mice, where it was found to be nontoxic and to have no detectable effect on the humoral response to soluble antigen.
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Antígenos CD4/genética , VIH/efectos de los fármacos , Inmunoglobulina G/genética , Inmunoglobulina M/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD4/metabolismo , Línea Celular , Femenino , Productos del Gen env/metabolismo , Células Gigantes/efectos de los fármacos , Proteínas gp160 de Envoltorio del VIH , Semivida , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Macaca fascicularis , Masculino , Ratones , Datos de Secuencia Molecular , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.
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Saccharomyces cerevisiae/genética , Transglutaminasas/genética , Clonación Molecular , Reactivos de Enlaces Cruzados/metabolismo , Fibrina/metabolismo , Fluorescencia , Humanos , Peso Molecular , Placenta/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Espectrofotometría Ultravioleta , Trombina/metabolismo , Transglutaminasas/biosíntesisRESUMEN
Antibody-like molecules consisting of the human CD 4 extracellular domain fused to human IgG1 heavy chain constant regions were genetically constructed and expressed in a BHK cell stable transfectant. Purified chimeric antibodies bound to HIV particles as it was shown by immuno electron microscopy, inhibited fusions of HIV-1-infected cells with uninfected cells, neutralized HIV-1, and were able to inhibit the spread of a cellular HIV-1 infection in CD 4+ cells. Plaque reduction assays with CD 4(+)-transfected Hela-cells showed a comparable inhibition of HIV-1 and HIV-2. Inhibitory functions were enhanced in the presence of complement. HIV-1- and HIV-2-infected CD 4+ cells were efficiently lysed by a slow, complement-dependent mechanism, whereas uninfected CD 4+ cells and HLA-DR+ cells were not affected.
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Antivirales , Antígenos CD4/farmacología , VIH-1/efectos de los fármacos , VIH/efectos de los fármacos , Cadenas Pesadas de Inmunoglobulina/farmacología , Proteínas Recombinantes de Fusión/farmacología , Animales , Antivirales/inmunología , Células Cultivadas , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Cobayas , Humanos , Cadenas gamma de Inmunoglobulina , Macrófagos/microbiología , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/inmunología , Ensayo de Placa ViralRESUMEN
The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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Reactivadores Enzimáticos , Placenta/análisis , Transglutaminasas/metabolismo , Fenómenos Químicos , Química Física , Femenino , Humanos , Embarazo , Conformación Proteica , Desnaturalización Proteica , Transglutaminasas/análisisRESUMEN
Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.