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Human Interferon (human + interferon)
Kinds of Human Interferon Selected AbstractsPurification, crystallization and preliminary crystallographic studies of the complex of interferon-,1 with its receptorACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2010Eugenia Magracheva Human interferon-,1 (IFN-,1Ins) and the extracellular domain of interferon-,1 receptor (IFN-,1R1) were expressed in Drosophila S2 cells and purified to homogeneity. Both IFN-,1Ins and interferon-,1 produced from Escherichia coli (IFN-,1Bac) were coupled with IFN-,1R1 at room temperature and the complexes were purified by gel filtration. Both complexes were crystallized; the crystals were flash-frozen at 100,K and diffraction data were collected to 2.16 and 2.1,Å, respectively. Although the IFN-,1Bac,IFN-,1R1 and IFN-,1Ins,IFN-,1R1 complexes differed only in the nature of the expression system used for the ligand, their crystallization conditions and crystal forms were quite different. A search for heavy-atom derivatives as well as molecular-replacement trials are in progress. [source] Increase of calnexin gene dosage boosts the secretion of heterologous proteins by Hansenula polymorphaFEMS YEAST RESEARCH, Issue 7 2007Jens Klabunde Abstract The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-, and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L,1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MF,1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha. [source] Antibody response to aggregated human interferon alpha2b in wild-type and transgenic immune tolerant mice depends on type and level of aggregationJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 5 2006Suzanne Hermeling Abstract The aim of this study was to determine the sensitivity of transgenic immune tolerant mice for the type and level of aggregation of recombinant human interferon alpha2b (rhIFN,2b). RhIFN,2b was aggregated by metal-catalyzed oxidation or by incubation at elevated temperature and various pHs. Native rhIFN,2b was mixed with oxidized rhIFN,2b at different ratios to obtain samples with different aggregation levels. The preparations were characterized by UV and fluorescence spectroscopy, gel permeation chromatography (GPC), dynamic light scattering (DLS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS,PAGE) and Western blotting, and ELISA. The immunogenicity was evaluated in wild-type mice and transgenic mice immune tolerant for hIFN,2. Sera were analyzed by ELISA for the presence of rhIFN,2b-specific antibodies. The oxidized and aged preparations widely differed regarding the level and nature of aggregates. All preparations containing aggregates increased the immune response in the wild-type mice as compared to native rhIFN,2b and were able to break the tolerance of the transgenic mice. The more native-like the conformation of the aggregated proteins, the more immunogenic the preparations were in the transgenic mice. The native-like aggregates prepared via metal catalysis induced a dose-dependent loss of tolerance in the transgenic mice. In conclusion, the transgenic mouse model can be used to screen rhIFN,2b formulations for low levels of immunogenic aggregates obtained under accelerated storage conditions. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1084,1096, 2006 [source] Effects of annealing lyophilized and spray-lyophilized formulations of recombinant human interferon-,JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2003Serena D. Webb Abstract The purpose of this study was to examine the effects of adsorption of recombinant human interferon-, (rhIFN-,) on ice surfaces and subsequent drying during processing by spray-lyophilization and lyophilization. Ice/liquid interfacial areas were manipulated by the freezing method as well as by the addition of an annealing step during lyophilization; that is, rhIFN-, adsorption was modified by the addition of nonionic surfactants. rhIFN-, was lyophilized or spray-lyophilized at a concentration of 1 mg/mL in 5% sucrose, 5% hydroxyethyl starch (HES),±,0.03% polysorbate 20 in 140 mM KCl, and 10 mM potassium phosphate, pH 7.5. After the samples were frozen, half were annealed on the lyophilizer shelf. Recovery of soluble protein was measured at intermediate points during processing. On drying, the secondary structure of rhIFN-, was determined by second-derivative infrared (IR) spectroscopy, specific surface areas (SSAs) were measured, scanning electron micrographs (SEM) were taken, and dissolution times were recorded. Adsorption of rhIFN-, to ice/liquid interfaces alone was not responsible for aggregation. Rather, drying was necessary to cause aggregation in lyophilized sucrose formulations. Addition of an annealing step to the lyophilization cycle resulted in more native-like secondary protein structure in the dried solid, eliminated cracking of the dried cakes, and suppressed both the formation of air/liquid interfaces and rhIFN-, aggregation on reconstitution. © 2003 Wiley-Liss, Inc. and the American pharmaceutical Association J Pharm Sci 92:715,729, 2003 [source] Determination of the human type I interferon receptor binding site on human interferon-,2 by cross saturation and an NMR-based model of the complexPROTEIN SCIENCE, Issue 11 2006Sabine R. Quadt-Akabayov Abstract Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFN,2 with R2-EC using multidimensional NMR techniques. NMR shows that IFN,2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFN,2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Å, and its well-defined orientation between IFN,2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand,receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules. [source] Profiling of N -glycosylation gene expression in CHO cell fed-batch culturesBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Danny Chee Furng Wong Abstract One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N -glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N -glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed-batch culture of a CHO cell line producing recombinant human interferon-, (IFN-,). Of the 24 N -glycosylation genes examined, 21 showed significant up- or down-regulation of gene expression as the fed-batch culture progressed from exponential, stationary and death phase. As the fed-batch culture progressed, there was also an increase in less sialylated IFN-, glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN-,. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed-batch strategy appears to need a 0.5,mM glutamine threshold to maintain similar N -glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible "bottlenecks" or "compromised" pathways in N -glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516,528. © 2010 Wiley Periodicals, Inc. [source] An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feedingBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Niki S.C. Wong Abstract Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N -glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-, (IFN-,). Galactose (±uridine), glucosamine (±uridine), and N -acetylmannosamine (ManNAc) (±cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-, sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex (,20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine,+,uridine and ManNAc,+,cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-, sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality. Biotechnol. Bioeng. 2010;107: 321,336. © 2010 Wiley Periodicals, Inc. [source] Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon ,-2AACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2009Vaheh Oganesyan Recombinant human interferon ,-2A (rhIFN-,-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/,) which targets several human interferon , subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I212121, with unit-cell parameters a = 134.82, b = 153.26, c = 163.49,Å. The diffraction of the crystals extended to 3.0,Å resolution. The asymmetric unit contained two Fab,rhIFN-,-2A complexes. This corresponded to a crystal volume per protein weight (VM) of 3.02,Å3,Da,1 and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity. [source] Heterologous Protein Production from the Inducible MET25 Promoter in Saccharomyces cerevisiaeBIOTECHNOLOGY PROGRESS, Issue 2 2005Steven P. Solow Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon ,, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer. [source] | |||||||||||||