Current Issue : July - September Volume : 2014 Issue Number : 3 Articles : 6 Articles
Background: Recombinant protein production is a process of great industrial interest, with products that range\nfrom pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in\nthe host organism, several methods have been developed over the years to optimize protein production. So far,\nthese trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been\nno attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of\nstandardized parts in novel synthetic circuits.\nResults: We present a novel self-regulatory protein production system that couples the control of recombinant\nprotein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the downregulation\nof recombinant protein expression through a stress-induced promoter. We used E. coli as the host\norganism, since it is widely used in recombinant processes. Our results show that the introduction of the selfregulatory\ncircuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield.\nTo further elucidate the dynamics of the system, we developed a computational model that is in agreement with\nthe observed experimental data, and provides insight on the interplay between protein solubility and yield.\nConclusion: Our work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves\nthe way for processes with reduced external control or monitoring needs. It demonstrates that the library of\nstandard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to\nbe successfully applied in practical problems of biotechnological significance. Finally, the development of a\npredictive model in conjunction with experimental validation facilitates a better understanding of the underlying\ndynamics and can be used as a guide to experimental design....
The present work aimed to study the biosimilarity of locally produced free IFN and innovated imported free one through evaluation of the antiviral activity of test interferons on different cell lines and related expression potentials of Mx protein producing gene. Evaluation of two alternative methods for evaluation the efficacy of antiviral interferon. Cytotoxicity of interferon was performed using different cell lines based on determination of residual live cells released lactate dehydrogenase (LDH) reactivity to MTT stain assay. Antiviral activity of locally produced and imported IFN was evaluated against vesicular stomatitis viruses (VSV). This assay was based on evaluation of the inhibitory activity to VSV grown on rh-IFN-α pretreated vero, MDBK and WISH cell lines. The potency of IFN was estimated by comparing inhibitory activity of the test interferons to standard one. Cytotoxicity evaluation results indicated that all types of test INF showed no toxicity to different cell lines. In the mean times tested IFN potency showed that locally produced one was not realized the international potency limits, while the imported one was accepted. Regarding the initiation of antiviral protein production was confirmed by detected Mx gene expressed in IFN pre treated cell lines and expression rate was related to cell type. Alternative way of evaluation of antiviral activity showed that there was a significant difference between local and imported products antiviral activity. Sensitivity of different cell lines showed that each tested product showed non significant difference (P>0.05) compared top each other. Different methodology could be established in the national regulatory authority (NRA) to evaluate interferon biosimillar bioactivity/ antiviral activity. Different cell lines could be used for evaluation of interferon antiviral activity in case of unavailability of recommended cell line....
In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly.\nMost applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and\nimprovement of a wide range of production platforms. No individual system is optimal for the production of all recombinant\nproteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four\nrecombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plantbased\nsystems over traditional fermenter-based expression platforms....
Glycosylation is a critical attribute of glycoprotein products as it has been shown that the type and degree of\r\nglycosylation can have a significant impact on the product efficacy and immunogenicity. In developing generic forms\r\nof glycoprotein based therapeutic products, it is necessary to characterize the glycosylation of these products to\r\nensure that it conforms with the original product as well as natural form of the product. In this study, we have focused\r\non the characterization of the N-linked glycans and, in less details, on the O-linked glycans found on Etanercept\r\n(EnbrelTM). Using a series of methodologies, we mapped the N-linked glycosylation sites in Etanercept and also\r\ndefined the types of glycan structures associated with each site. Separately, we also determined the extent of\r\nEtanercept O-glycosylation and the type of O-linked glycans....
rHuEPO plays a central role as chemicals for the treatment of many diseases. Due to its short half-life, the main aim for\nthis pharmacokinetic study is to investigate a newly developed PEG-rHuEPO with large molecular weight in SD rats. After a\nsingle intramuscular administration of different doses of 125I-PEG-rHuEPO, pharmacokinetic parameters, tissue distribution,\nand excretion were analyzed. In in vivo half-life time measured after 125I-PEG-rHuEPO administration at the doses of 1, 2, and\n3�µg/kg, t1/2a was 1.90, 1.19, and 2.50 hours, respectively, whereas t1/2�Ÿ was 22.37, 26.21, and 20.92 hours, respectively; at 8, 24,\nand 48 hours after intramuscular administration, PEG-rHuEPO was distributed to all of the examined tissues, however, with high\nconcentrations of radioactivity, only in plasma, blood, muscle at the administration site, and bone marrow. Following a 2 �µg/kg\nsingle intramuscular administration, approximately 21% of the radiolabeled dose was recovered after almost seven days of study.\nUrine was the major route of excretion; 20% of the administered dose was recovered in the urine, while excretion in the feces was\nless than 1.4%. Therefore, this PEG-rHuEPO has potential to be clinically used and could reduce frequency of injection....
Background: The potential benefit of adding recombinant human luteinizing hormone (r-hLH) to recombinant\nhuman follicle-stimulating hormone (r-hFSH) during ovarian stimulation is a subject of debate, although there is\nevidence that it may benefit certain subpopulations, e.g. poor responders.\nMethods: A systematic review and a meta-analysis were performed. Three databases (MEDLINE, Embase and\nCENTRAL) were searched (from 1990 to 2011). Prospective, parallel-, comparative-group randomized controlled trials\n(RCTs) in women aged 18ââ?¬â??45 years undergoing in vitro fertilization, intracytoplasmic sperm injection or both,\ntreated with gonadotrophin-releasing hormone analogues and r-hFSH plus r-hLH or r-hFSH alone were included.\nThe co-primary endpoints were number of oocytes retrieved and clinical pregnancy rate. Analyses were conducted\nfor the overall population and for prospectively identified patient subgroups, including patients with poor ovarian\nresponse (POR).\nResults: In total, 40 RCTs (6443 patients) were included in the analysis. Data on the number of oocytes retrieved\nwere reported in 41 studies and imputed in two studies. Therefore, data were available from 43 studies (r-hFSH plus\nr-hLH, n = 3113; r-hFSH, n = 3228) in the intention-to-treat (ITT) population (all randomly allocated patients, including\nimputed data). Overall, no significant difference in the number of oocytes retrieved was found between the r-hFSH\nplus r-hLH and r-hFSH groups (weighted mean difference -0.03; 95% confidence interval [CI] -0.41 to 0.34). However,\nin poor responders, significantly more oocytes were retrieved with r-hFSH plus r-hLH versus r-hFSH alone (n =\n1077; weighted mean difference +0.75 oocytes; 95% CI 0.14ââ?¬â??1.36). Significantly higher clinical pregnancy rates were\nobserved with r-hFSH plus r-hLH versus r-hFSH alone in the overall population analysed in this review (risk ratio [RR]\n1.09; 95% CI 1.01ââ?¬â??1.18) and in poor responders (n = 1179; RR 1.30; 95% CI 1.01ââ?¬â??1.67; ITT population); the observed\ndifference was more pronounced in poor responders....
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