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Recombinant Protein Expression (recombinant + protein_expression)
Selected AbstractsComparison of Growth and Recombinant Protein Expression in Two Different Insect Cell Lines in Attached and Suspension CultureBIOTECHNOLOGY PROGRESS, Issue 4 2001R. A. Taticek Culture conditions required for obtaining maximum recombinant protein concentrations from two cell lines, Spodoptera frugiperda (IPL,-Sf21-AE) and Trichoplusia ni (Tn 5,-1,4), were determined in this work. Conditions studied include mode of culture (suspended vs attached), agitation rates, inoculum sizes, cell concentration at the time of infection, and various serum-free media (SFM). Results were compared with the performance of attached cultures in TnM-FH with 10% fetal bovine serum. Growth rates in the different culture media tested were similar, but the cell numbers achieved (i.e., yield) improved 2 to 2.7-fold in SFM over cultures in TnM-FH. Agitation rates of 150,160 rpm were necessary for maximum growth of suspended Tn 5,-1,4 cells compared to 125,150 rpm for Sf-21 cells. An inoculum size of 5 × 105 cells/mL gave good growth rates and optimum biomass yields for both cell lines. Cultures of both cell lines were infected with viruses encoding for ,-galactosidase or human secreted alkaline phosphatase (seAP). Protein expression in TnM-FH in attached culture showed that Tn 5,-1,4 cells are 2,4.5 times more productive on a per cell basis than Sf-21 cells grown under similar conditions. Production of ,-galactosidase in Sf-21 cells increased 50% in suspension cultures with SFM compared to attached cultures in TnM-FH, but seAP expression was essentially unchanged by culture techniques. The Tn 5,-1,4 cells produced 2.6,4.4 and 2.7,3 times more ,-galactosidase and seAP, respectively, in SFM in suspension compared to Sf-21 cells. EX-CELL 401 and Sf900-II were formulated as optimized SFM for Sf cell lines. However, in Sf-21 cultures EX-CELL 400 performed better than the other two media, as it increased the ,-galactosidase yield up to 25%. Surprisingly, EX-CELL 401 was the best medium for the production of ,-galactosidase by Tn 5,-1,4 cells, resulting in 25% and 69% higher volumetric and specific yields, respectively, compared to EX-CELL 405 which was formulated for this specific cell line. These results show that even when culture media are designed for maximal growth of a specific cell line, other media may provide the best conditions for protein production. [source] Recombinant protein expression and solubility screening in Escherichia coli: a comparative studyACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006Nick S. Berrow Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His6 -tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore `expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization. [source] Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplastTHE PLANT JOURNAL, Issue 6 2002Scott Franklin Summary Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5,- and 3,-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5,- and 3,-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate ,80-fold more GFP than GFPncb -transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5,- and 3,-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression. [source] Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technologyBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Malcolm L. Kennard Abstract In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO-S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO-S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO-S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO-S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526,539 © 2009 Wiley Periodicals, Inc. [source] Production of recombinant proteins using multiple-copy gene integration in high-cell-density fermentations of Ralstonia eutrophaBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2003Sriram Srinivasan Abstract We have previously reported the development of a novel protein expression system based on Ralstonia eutropha. In this study we report on the influence of gene copynumber on recombinant protein expression in R. eutropha. We compare recombinant gene stability and expression levels of chromosomal integration with a plasmid-based expression system. Single, double, and triple copies of a gene encoding organophosphohydrolase (OPH), an enzyme prone to inclusion-body formation in E. coli, were integrated into the R. eutropha chromosome. A linear increase between the concentration of soluble, active OPH and gene copynumber was found. Using a triple-copy integrant, we were able to produce approximately 4.3 g/L of OPH in a high-cell-density fermentation. This represents the highest titer reported to date for this enzyme, and is approximately 30 times greater than expression levels reported in E. coli. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 114,120, 2003. [source] Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2002Idsada Lengwehasatit Abstract Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 719,730, 2002. [source] Insights into the Central Metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4(Tn -5) Insect Cells by Radiolabeling StudiesBIOTECHNOLOGY PROGRESS, Issue 1 2005Chouki Benslimane The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia niBTI-Tn-5B1 - 4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed. [source] Rapid Matrix-Assisted Refolding of Histidine-Tagged ProteinsCHEMBIOCHEM, Issue 5 2009Tetyana Dashivets Abstract Matrix refolded: The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in-solution, this parallelized, matrix-assisted approach allows the refolding of various proteins in a fast and efficient manner. The formation of inclusion bodies (IBs),amorphous aggregates of misfolded insoluble protein,during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His6 -tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence,it allows refolding at up to 1000-fold higher protein concentration than folding in-solution. [source] | |||||||||||||