Home About us Contact
|
Home About us Contact | ||
|
|
||
Promoter System (promoter + system)
Selected AbstractsProduction of a Secreted Glycoprotein from an Inducible Promoter System in a Perfusion BioreactorBIOTECHNOLOGY PROGRESS, Issue 5 2004Matthew L. Lipscomb The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated. [source] Arabinose-Induction of lac -Derived Promoter Systems for Penicillin Acylase Production in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2006Niju Narayanan Arabinose was shown to serve as an effective inducer for induction of the lac -derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-,- d -thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by thetrc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac -derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LLpac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression. [source] Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databasesELECTROPHORESIS, Issue 1-2 2003Karin Dürrschmid Abstract The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3,10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response. [source] Doc-mediated cell killing in Shigella flexneri using a C1/LacI controlled expression systemFEMS MICROBIOLOGY LETTERS, Issue 2 2002David A. Schofield Abstract In this report we describe the development of a highly stringent and dually regulated promoter system for Shigella flexneri. Dual regulation was provided by utilizing a promoter susceptible to control by the bacteriophage P1 temperature-sensitive C1 repressor that in turn was under the transcriptional control of LacI. The level of induction/repression ratios observed was up to 3700-fold in S. flexneri. The general utility of this promoter system was evaluated by demonstrating that the bacteriophage P1 post-segregational killer protein Doc mediates a bactericidal effect in S. flexneri. This represents the first report of Doc (death on curing)-mediated killing in this Gram-negative species. [source] Transcription of rat TRPV1 utilizes a dual promoter system that is positively regulated by nerve growth factorJOURNAL OF NEUROCHEMISTRY, Issue 1 2007Qing Xue Abstract The capsaicin receptor, also known as ,transient receptor potential vanilloid receptor subtype 1' (TRPV1, VR1), is an ion channel subunit expressed in primary afferent nociceptors, which plays a critical role in pain transduction and thermal hyperalgesia. Increases in nociceptor TRPV1 mRNA and protein are associated with tissue injury,inflammation. As little is understood about what controls TRPV1 RNA transcription in nociceptors, we functionally characterized the upstream portion of the rat TRPV1 gene. Two functional rTRPV1 promoter regions and their transcription initiation sites were identified. Although both promoter regions directed transcriptional activity in nerve growth factor (NGF) treated rat sensory neurons, the upstream Core promoter was the most active in cultures enriched in sensory neurons. Because NGF is a key modulator of inflammatory pain, we examined the effect of NGF on rTRPV1 transcription in PC12 cells. NGF positively regulated transcriptional activity of both rTRPV1 promoter regions in PC12 cells. We propose that the upstream regulatory region of the rTRPV1 gene is composed of a dual promoter system that is regulated by NGF. These findings support the hypothesis that NGF produced under conditions of tissue injury and/or inflammation directs an increase of TRPV1 expression in nociceptors in part through a transcription-dependent mechanism. [source] In vivo electroporation and ubiquitin promoter , a protocol for sustained gene expression in the lungTHE JOURNAL OF GENE MEDICINE, Issue 7 2006Amiq Gazdhar Abstract Background Gene therapy applications require safe and efficient methods for gene transfer. Present methods are restricted by low efficiency and short duration of transgene expression. In vivo electroporation, a physical method of gene transfer, has evolved as an efficient method in recent years. We present a protocol involving electroporation combined with a long-acting promoter system for gene transfer to the lung. Methods The study was designed to evaluate electroporation-mediated gene transfer to the lung and to analyze a promoter system that allows prolonged transgene expression. A volume of 250 µl of purified plasmid DNA suspended in water was instilled into the left lung of anesthetized rats, followed by left thoracotomy and electroporation of the exposed left lung. Plasmids pCiKlux and pUblux expressing luciferase under the control of the cytomegalovirus immediate-early promoter/enhancer (CMV-IEPE) or human polyubiquitin c (Ubc) promoter were used. Electroporation conditions were optimized with four pulses (200 V/cm, 20 ms at 1 Hz) using flat plate electrodes. The animals were sacrificed at different time points up to day 40, after gene transfer. Gene expression was detected and quantified by bioluminescent reporter imaging (BLI) and relative light units per milligram of protein (RLU/mg) was measured by luminometer for p.Pyralis luciferase and immunohistochemistry, using an anti-luciferase antibody. Results Gene expression with the CMV-IEPE promoter was highest 24 h after gene transfer (2932 ± 249.4 relative light units (RLU)/mg of total lung protein) and returned to baseline by day 3 (382 ± 318 RLU/mg of total lung protein); at day 5 no expression was detected, whereas gene expression under the Ubc promoter was detected up to day 40 (1989 ± 710 RLU/mg of total lung protein) with a peak at day 20 (2821 ± 2092 RLU/mg of total lung protein). Arterial blood gas (PaO2), histological assessment and cytokine measurements showed no significant toxicity neither at day 1 nor at day 40. Conclusions These results provide evidence that in vivo electroporation is a safe and effective tool for non-viral gene delivery to the lungs. If this method is used in combination with a long-acting promoter system, sustained transgene expression can be achieved. Copyright © 2006 John Wiley & Sons, Ltd. [source] Reduction of oxidative changes in human spermatozoa by exogenous gangliosidesANDROLOGIA, Issue 1 2005M. Gavella Summary The effect of exogenous gangliosides, the sialic acid-containing glycosphingolipids, on oxidative changes in human spermatozoa was investigated. The incorporation of disialogangliosides or trisialogangliosides (GD1b and GT1b, respectively) into the iron/ascorbate promoter system for induction of lipid peroxidation decreased the release of malondialdehyde (MDA) from peroxidizing spermatozoa. The application of monosialogangliosides and disialogangliosides (GM1 and GD1a, respectively) did not have any effect under identical experimental conditions. GT1b, at a micromolar concentration, significantly inhibited the production of MDA, a breakdown product of lipid peroxide decomposition in spermatozoa of normozoospermic infertile men (P < 0.001; n = 51). An enhanced generation of MDA exhibited by the sperm population from the low-density Percoll fraction containing defective and/or immature spermatozoa was significantly reduced in the presence of GT1b. These results and the experiments on the influence of iron-chelating agent ethylenediamine tetraacetic acid (EDTA) as well as ferrous ion concentration itself on lipid peroxidation support the hypothesis that the protective effect of ganglioside against MDA generation could be the result of its chelating activity. Furthermore, superoxide anion release of phorbol myristate acetate-stimulated spermatozoa was significantly reduced in the presence of 50 and 100 ,mol l,1 GD1b (P < 0.05) and GT1b (P < 0.005). The inhibitory effect of 100 ,mol l,1 GT1b on spermatozoa from infertile normozoospermic men was statistically significant (P < 0.001; n = 21) and did not depend on the initial superoxide anion production. In conclusion, the protective action of GD1b and GT1b could be related to both scavenging of free radicals and metal-chelating properties, which might have relevance in the protection against oxidation-induced processes in human spermatozoa. [source] Acetate-inducible protein overexpression from the glnAp2 promoter of Escherichia coliBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2001William R. Farmer Abstract The Ntr regulon in Escherichia coli has previously been engineered to control the expression of a heterologous metabolic pathway. In this study, we reengineered the same system for protein production. In the absence of NRII (glnL gene product), we showed that glnAp2 can be an effective promoter for protein production that is inducible by exogenous acetate, but both the induction ratio and the range of modulation are low. To deal with this issue, we inactivated phosphotransacetylase (pta gene product), which disrupts the acetate pathway and denies the cell the ability to synthesize acetate. With this additional modification, gene expression from glnAp2 can be controlled by directly adding acetate into the growth medium. Using a lacZ reporter fusion, we found that glnAp2 induction was modulatable over a range of potassium acetate concentrations, and the induction/noninduction ratio increased to 77 in the absence of pta. The extracellular acetate required for maximal induction is lower than the concentration that causes toxicity, and thus growth inhibition by acetate addition was not a matter of concern. Furthermore, compared to the Ptac promoter, overexpression of a model protein using the modified glnAp2 promoter system did not cause significant growth inhibition, although a higher level of protein expression was achieved. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 504,509, 2001. [source] Arabinose-Induction of lac -Derived Promoter Systems for Penicillin Acylase Production in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2006Niju Narayanan Arabinose was shown to serve as an effective inducer for induction of the lac -derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-,- d -thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by thetrc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac -derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LLpac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression. [source] Transient Effects of Overexpressing Anthranilate Synthase , and , Subunits in Catharanthusroseus Hairy RootsBIOTECHNOLOGY PROGRESS, Issue 5 2005Christie A. M. Peebles Catharanthus roseus produces two economically valuable anticancer drugs, vinblastine and vincristine. These drugs are members of the terpenoid indole alkaloids and accumulate in small quantities within the plant; thus these two drugs are expensive to produce. Metabolic engineering efforts have focused on increasing the alkaloids in this pathway through various means such as elicitation, precursor feeding, and gene overexpression. Recently we successfully expressed Arabidopsis genes encoding a feedback-insensitive anthranilate synthase , subunit under the control of the glucocorticoid-inducible promoter system and the anthranilate synthase , subunit under the control of a constitutive promoter in C. roseus hairy roots. In this work we look at the transient behaviors of terpenoid indole alkaloids over a 72 h induction period in late exponential growth phase cultures. Upon induction, the tryptophan, tryptamine, and ajmalicine pools accumulated over 72 h. In contrast, the lochnericine, hörhammericine, and tabersonine pools decreased and leveled out over the 72 h induction period. Visible changes within the individual compounds usually took from 4 to 12 h. [source] Arabinose-Induction of lac -Derived Promoter Systems for Penicillin Acylase Production in Escherichia coliBIOTECHNOLOGY PROGRESS, Issue 3 2006Niju Narayanan Arabinose was shown to serve as an effective inducer for induction of the lac -derived promoters in Escherichia coli using penicillin acylase (PAC) as a model protein. Upon the induction with a conventional inducer, isopropyl-,- d -thiogalactopyranoside (IPTG), for pac overexpression, which is regulated by thetrc or (DE3)/T7 promoter, the production of PAC was limited by the accumulation of PAC precursors (proPAC) as inclusion bodies. Negative cellular responses, such as growth inhibition and cell lysis, were frequently observed, resulting in a low pac expression level and poor culture performance. Interestingly, these technical hurdles can be overcome simply through the use of arabinose as an inducer. The results indicate that arabinose not only induced the lac -derived promoter systems (i.e., trc and (DE3)/T7) for pac (or LLpac) overexpression but also facilitated the posttranslational processing of proPAC for maturation. However, the arabinose-inducibility appears to be host-dependent and becomes less observable in the strains with a mutation in the ara operon. The arabinose-inducibility was also investigated in the expression system with the coexistence of the trc promoter system regulating pac expression and another arabinose-inducible promoter system of araB regulating degP coexpression. [source] | ||||||||||