Knockout of caspase-7 gene improves the expression of recombinant protein in CHO cell line through the cell cycle arrest in G2/M phase

BACKGROUND: Chinese hamster ovary cell line has been used routinely as a bioproduction factory of numerous biopharmaceuticals. So far, various engineering strategies have been recruited to improve the production efficiency of this cell line such as apoptosis engineering. Previously, it is reported that the caspase-7 deficiency in CHO cells reduces the cell proliferation rate. But the effect of this reduction on the CHO cell productivity remained unclear. Hence, in the study at hand the effect of caspase-7 deficiency was assessed on the cell growth, viability and protein expression. In addition, the enzymatic activity of caspase-3 was investigated in the absence of caspase-7. RESULTS: Findings showed that in the absence of caspase-7, both cell growth and cell viability were decreased. Cell cycle analysis illustrated that the CHO knockout (CHO-KO) cells experienced a cell cycle arrest in G2/M phase. This cell cycle arrest resulted in a 1.7-fold increase in the expression of luciferase in CHO-KO cells compared to parenteral cells. Furthermore, in the apoptotic situation the enzymatic activity of caspase-3 in CHO-KO cells was approximately 3 times more than CHO-K1 cells. CONCLUSIONS: These findings represented that; however, caspase-7 deficiency reduces the cell proliferation rate but the resulted cell cycle arrest leads to the enhancement of recombinant protein expression. Moreover, increasing in the caspase-3 enzymatic activity compensates the absence of caspase-7 in the caspase cascade of apoptosis.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Plastid transformation: advances and challenges for its implementation in agricultural crops

Chloroplast biotechnology has emerged as a promissory platform for the development of modified plants to express products aimed mainly at the pharmaceutical, agricultural, and energy industries. This technology's high value is due to its high capacity for the mass production of proteins. Moreover, the interest in chloroplasts has increased because of the possibility of expressing multiple genes in a single transformation event without the risk of epigenetic effects. Although this technology solves several problems caused by nuclear genetic engineering, such as turning plants into safe bio-factories, some issues must still be addressed in relation to the optimization of regulatory regions for efficient gene expression, cereal transformation, gene expression in non-green tissues, and low transformation efficiency. In this article, we provide information on the transformation of plastids and discuss the most recent achievements in chloroplast bioengineering and its impact on the biopharmaceutical and agricultural industries; we also discuss new tools that can be used to solve current challenges for their successful establishment in recalcitrant crops such as monocots.

Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l -1 in comparison with the 284 mg l -1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.

Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Expression of a recombinant protein, A2 family, from Leishmania infantum (Jaboticabal strain) and its evaluation in Canine Visceral Leishmaniasis serological test / Expressão de uma proteína recombinante, da família A2, de Leishmania infantum (amostra Jaboticabal) e sua avaliação no teste sorológico da Leishmaniose Visceral Canina

This study aimed to express a recombinant A2 family protein of Leishmania chagasi, Jaboticabal strain; test this protein as an antigen in serological assays; and investigate its antigenicity and immunogenicity. A protein coded by an allele of the A2 gene isolated from L. chagasi was expressed in three different strains of Escherichia coli. We used 29 sera samples from Leishmune-vaccinated dogs, 482 sera samples from dogs from endemic areas (positive controls), and 170 sera samples from dogs from non-endemic areas (negative controls) in ELISA tests using soluble Leishmaniaantigen (SLA) and His-A2 as antigen. Expressed proteins showed, by western blotting, the expression of an 11 KDa protein. Sixty-three percent (303/482) of the samples from endemic areas were positive by ELISA His-A2, whereas 93.1% (27/29) of Leishmune®-vaccinated animals were negative by His-A2-ELISA. Anti-A2 antibodies from mice inoculated with the A2 protein were detected in slides containing amastigote forms, but not in slides containing promastigote forms. The A2 recombinant protein from L. chagasi may be a useful tool in the diagnosis of CVL, and further tests regarding the infection stage and the specie of parasite at which the dogs are sampled should provide a better understanding of our results.

Este estudo teve como objetivos expressar uma proteína recombinante da família A2 de Leishmania chagasi, amostra de Jaboticabal-SP; testar essa proteína como antígeno em testes sorológicos; e investigar a antigenicidade e imunogenicidade dessa proteína. Uma proteína codificada por um alelo do gene A2 isolado de L. chagasi foi expressa em três diferentes amostras de Escherichia coli. Foram utilizadas 29 amostras de soro de cães vacinados com Leishmune, 482 amostras de soro de cães de áreas endêmicas (controles positivos), e 170 amostras de soro de cães de áreas não-endêmicas (controles negativos) no ELISA-teste utilizando-se antígeno solúvel total de Leishmania (AST) e His-A2 como antígenos. As proteínas expressas, detectadas pelo western blotting, mostraram a expressão de uma proteína de 11 KDa. Sessenta e três por cento (303/482) das amostras de áreas endêmicas foram positivas pelo ELISA-teste, utilizando-se antígeno His-A2; e 93,1% (27/29) dos animais vacinados com a Leishmune foram negativos. Anticorpos anti-A2 de camundongos inoculados com a proteína A2 foram detectados em lâminas contendo formas amastigotas, enquanto em lâminas contendo formas promastigotas não houve detecção de anticorpos anti-A2. A proteína recombinante A2 pode ser uma ferramenta útil no diagnóstico da LVC, e maiores estudos sobre o estágio de infecção e a espécie de parasita dos cães amostrados devem prover melhor entendimento dos resultados encontrados.

The Effectiveness of Recombinant Human Thyroid-Stimulating Hormone versus Thyroid Hormone Withdrawal Prior to Radioiodine Remnant Ablation in Thyroid Cancer: A Meta-Analysis of Randomized Controlled Trials

We evaluated the efficacy of recombinant human thyroid-stimulating hormone (rhTSH) versus thyroid hormone withdrawal (THW) prior to radioiodine remnant ablation (RRA) in thyroid cancer. A systematic search of MEDLINE, EMBASE, the Cochrane Library, and SCOPUS was performed. Randomized controlled trials that compared ablation success between rhTSH and THW at 6 to 12 months following RRA were included in this study. Six trials with a total of 1,660 patients were included. When ablation success was defined as a thyroglobulin (Tg) cutoff of 1 ng/mL (risk ratio, 0.99; 95% confidence interval, 0.96-1.03) or a Tg cutoff of 1 ng/mL plus imaging modality (RR 0.97; 0.90-1.05), the results of rhTSH and THW were similar. There were no significant differences when ablation success was defined as a Tg cutoff of 2 ng/mL (RR 1.03; 0.95-1.11) or a Tg cutoff of 2 ng/mL plus imaging modality (RR 1.02; 0.95-1.09). When a negative 131I-whole body scan was used solely as the definition of ablation success, the effects of rhTSH and THW were not significantly different (RR 0.97; 0.93-1.02). Therefore, ablation success rates are comparable when RRA is prepared by either rhTSH or THW.

Development of Enzyme-Linked Immunosorbent Assays Using 2 Truncated ORF2 Proteins for Detection of IgG Antibodies Against Hepatitis E Virus

BACKGROUND: Without appropriate culture systems for hepatitis E virus (HEV), sufficient natural viral proteins are difficult to generate for use in serological tests. Therefore, it is important to produce large amounts of HEV recombinant proteins in an economical way. The present study developed ELISAs using 2 truncated forms of the HEV open reading frame (ORF) 2 protein in order to detect anti-HEV IgG in serum samples. METHODS: Two truncated forms of the ORF2 protein were expressed in Escherichia coli and were purified by Ni2+-chelate-affinity chromatography (Qiagen, Germany). Two ELISAs were developed using these proteins and were compared with DIA.PRO HEV IgG ELISA kit (DIA.PRO. Italy) in 220 serum samples. RESULTS: High yields of the target proteins were obtained through codon optimization. The concentration and purity of the proteins were improved with Amicon filters (EMD Millipore, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis of the resultant proteins showed a protein band of approximately 60 kDa corresponding to ORF2.1 (amino acids 112-660) and a protein band of approximately 55 kDa corresponding to ORF2.2 (amino acids 112-607). Positive agreement, negative agreement, and concordance of the 2 in-house ELISAs compared with DIA.PRO HEV IgG ELISA kit were 87%, 99.5%, and 98.1%, respectively (kappa=0.899, P=0.625). CONCLUSIONS: The newly developed ELISAs are useful for detecting anti-HEV IgG in serum samples and are highly concordant with DIA.PRO HEV IgG ELISA kit.

Effects of Antioxidant on Reduction of Hindlimb Muscle Atrophy Induced by Cisplatin in Rats

PURPOSE: The purpose of this study was to examine the effects of Cu/Zn SOD on reduction of hindlimb muscular atrophy induced by cisplatin in rats. METHODS: Forty-two rats were assigned to three groups; control group, Cisplatin (CDDP) group and cisplatin with Cu/Zn SOD (CDDP-SOD) group. At day 35 hindlimb muscles were dissected. Food intake, activity, withdrawal threshold, muscle weight, and Type I, II fiber cross-sectional area (CSA) of dissected muscles were measured. Relative SOD activity and expression of MHC and phosphorylated Akt, ERK were measured after dissection. RESULTS: Muscle weight and Type I, II fiber CSA of hindlimb muscles in the CDDP group were significantly less than the control group. Muscle weight and Type I, II fiber CSA of hindlimb muscles, food intake, activity, and withdrawal thresholds of the CDDP-SOD group were significantly greater than the CDDP group. There were no significant differences in relative SOD activities of hindlimb muscles between the CDDP-SOD and CDDP groups. MHC expression and phosphorylated Akt, ERK of hindlimb muscles in the CDDP-SOD group were significantly greater than the CDDP group. CONCLUSION: Cu/Zn SOD attenuates hindlimb muscular atrophy induced by cisplatin through increased food intake and activity. Increment of phosphorylated Akt, ERK may relate to attenuation of hindlimb muscular atrophy.

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space (i.e. 4 hrs post induction).

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.

Expression enhancement in trastuzumab therapeutic monoclonal antibody production using genomic amplification with methotrexate

Trastuzumab [Herceptin] is a humanized monoclonal antibody [mAb] which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2[+] tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase [DHFR]. It is usually accepted that DHFR gene can be amplified in DHFR CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR

Intrathecal Gabapentin Increases Interleukin-10 Expression and Inhibits Pro-Inflammatory Cytokine in a Rat Model of Neuropathic Pain

We examined the possible anti-inflammatory mechanisms of gabapentin in the attenuation of neuropathic pain and the interaction between the anti-allodynic effects of gabapentin and interleukin-10 (IL-10) expression in a rat model of neuropathic pain. The anti-allodynic effect of intrathecal gabapentin was examined over a 7-day period. The anti-allodynic effects of IL-10 was measured, and the effects of anti-IL-10 antibody on the gabapentin were assessed. On day 7, the concentrations of pro-inflammatory cytokines and IL-10 were measured. Gabapentin produced an anti-allodynic effect over the 7-day period, reducing the expression of pro-inflammatory cytokines but increasing the expression of IL-10 (TNF-alpha, 316.0 +/- 69.7 pg/mL vs 88.8 +/- 24.4 pg/mL; IL-1beta, 1,212.9 +/- 104.5 vs 577.4 +/- 97.1 pg/mL; IL-6, 254.0 +/- 64.8 pg/mL vs 125.5 +/- 44.1 pg/mL; IL-10, 532.1 +/- 78.7 pg/mL vs 918.9 +/- 63.1 pg/mL). The suppressive effect of gabapentin on pro-inflammatory cytokine expression was partially blocked by the anti-IL-10 antibody. Expression of pro-inflammatory cytokines was significantly attenuated by daily injections of IL-10. The anti-allodynic effects of gabapentin may be caused by upregulation of IL-10 expression in the spinal cord, which leads to inhibition of the expression of pro-inflammatory cytokines in the spinal cords.

Improvement of the Diagnostic Sensitivity of Scrub Typhus Using a Mixture of Recombinant Antigens Derived from Orientia tsutsugamushi Serotypes

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.

Changes in Anti-Group A Rotavirus Antibody Seroprevalence and Levels in the Western Gyeongnam Province of Korea Over 16 Years

To observe how anti-group A rotavirus antibody seropositivity rates and levels have changed in the western region of Gyeongnam Province, 2,030 serum samples collected at four collection periods (1989-1990, 1994-1995, 1999-2000, and 2004-2005) were tested by Enzyme-Linked Immunosorbent Assay for IgG, and IgA antibodies reacting to recombinant VP6 protein. The seroprevalences exhibit no regular patterns over a 16-yr period. For all four collection periods, the anti-rVP6 IgG levels rose steadily during the first 5 months of life, after which they remained high. However, the 2-9 yr and 10-39 yr groups had significantly higher IgG levels in 1999-2000 and 2004-2005, respectively, than in the other collection periods. The 1-5 mo, 40- > or = 60 yr, and 4-29 yr groups had significantly higher IgA levels in 1989-1990, 1999-2000, and 2004-2005, respectively. The 4 yr (25.0%), 5-9 yr (18.8%), 10-14 yr (41.1%), 20-29 yr (35.0%), and 30-39 yr (20.0%) groups in 2004-2005 had significant higher IgA seropositivity rate compared to the other three collection periods. These observations suggest that in the western region of Gyeongnam Province since the late 1990s, rotavirus reinfection has occurred more frequently than previously, with all ages being at risk.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.