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Rate Limiting Step (rate + limiting_step)
Selected AbstractsEnhancing plant growth and fiber production by silencing GA 2-oxidasePLANT BIOTECHNOLOGY JOURNAL, Issue 4 2010Jonathan Dayan Summary Enhancing plant height and growth rates is a principal objective of the fiber, pulp, wood and biomass product industries. Many biotechnological systems have been established to advance that task with emphasis on increasing the concentration of the plant hormone gibberellin, or on its signalling. In this respect, the most studied gibberellin biosynthesis enzyme is the GA 20-oxidase which catalyses the rate limiting step of the pathway. Overexpression of the gene resulted in an excessively high activity of the gibberellin deactivating enzyme, GA 2-oxidase. Consequently, this feedback regulation limits the intended outcome. We assume that silencing GA 2-oxidase transcription would abolish this antithetical effect, thereby allowing greater gibberellin accumulation. Here, we show that silencing the gibberellin deactivating enzyme in tobacco model plants results in a dramatic improvement of their growth characteristics, compared with the wild type and GA 20-oxidase over-expressing plants. Moreover, the number of xylem fiber cells in the silenced lines exceeded that of GA 20-oxidase over-expressing plants, potentially, making GA 2-oxidase silencing more profitable for the wood and fiber industries. Interestingly, crossing GA 20-oxidase over-expressing plants with GA 2-oxidase silenced plants did not yield consequential additive effects. Our findings unveil the benefits of silencing GA 2-oxidase to substantially increase tobacco growth and fiber production, which suggest using this approach in cultivated forest plantations and industrial herbaceous plants, worldwide. [source] Low-temperature photosynthetic performance of a C4 grass and a co-occurring C3 grass native to high latitudesPLANT CELL & ENVIRONMENT, Issue 7 2004D. S. KUBIEN ABSTRACT The photosynthetic performance of C4 plants is generally inferior to that of C3 species at low temperatures, but the reasons for this are unclear. The present study investigated the hypothesis that the capacity of Rubisco, which largely reflects Rubisco content, limits C4 photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco between 5 and 35 °C were measured to examine the nature of the low-temperature photosynthetic performance of the co-occurring high latitude grasses, Muhlenbergia glomerata (C4) and Calamogrostis canadensis (C3). Plants were grown under cool (14/10 °C) and warm (26/22 °C) temperature regimes to examine whether acclimation to cool temperature alters patterns of photosynthetic limitation. Low-temperature acclimation reduced photosynthetic rates in both species. The catalytic site concentration of Rubisco was approximately 5.0 and 20 µmol m,2 in M. glomerata and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates below the thermal optimum exceeded what was necessary to support photosynthesis. In warm-grown C. canadensis, the photosynthesis rate below 15 °C was unaffected by a 90% reduction in O2 content, indicating photosynthetic capacity was limited by the capacity of Pi -regeneration. By contrast, the rate of photosynthesis in C. canadensis plants grown at the cooler temperatures was stimulated 20,30% by O2 reduction, indicating the Pi -regeneration limitation was removed during low-temperature acclimation. In M. glomerata, in vitro Rubisco activity and gross CO2 assimilation rate were equivalent below 25 °C, indicating that the capacity of the enzyme is a major rate limiting step during C4 photosynthesis at cool temperatures. [source] Effect of inducible co-overexpression of protein disulfide isomerase and endoplasmic reticulum oxidoreductase on the specific antibody productivity of recombinant Chinese hamster ovary cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Chaya Mohan Abstract To enhance specific antibody (Ab) productivity (qAb) of recombinant Chinese hamster ovary (rCHO) cells, post-translational limitations in the endoplasmic reticulum during antibody production should be relieved. Previously, we reported that overexpression of protein disulfide isomerase (PDI), which catalyzes disulfide bond exchanges and assists in protein folding of newly synthesized proteins, enhanced qAb of rCHO cells by about 27% (Mohan et al., 2007, Biotechnol Bioeng 98:611,615) . Since the rate limiting step in disulfide bond formation is found to be the regeneration of oxidized PDI, the oxidation state of PDI, as well as the amount of PDI, might be important. Endoplasmic reticulum oxidoreductase (ERO1L) maintains PDI in an oxidized state so that disulfide bond formation occurs. Here, PDI and its helper protein, ERO1L were overexpressed in rCHO cells producing an Ab in an attempt to ease the bottleneck in disulfide bond formation, and hence, Ab folding and secretion. Transient expression of ERO1L alone and with PDI resulted in enhanced qAb by 37% and 55%, respectively. In contrast, under stable inducible co-overexpression of PDI and ERO1L, the qAb was unaffected or negatively affected by varying degrees, depending on the individual expression levels of these genes. In stable clones with altered oxidation state of PDI due to co-overexpression of PDI and ERO1L, secretion of Ab was hindered and PDI-associated retention of Ab was seen in the cells. Under transient gene expression, secretion of Ab was not compromised. The data presented here suggests a possible mechanism of PDI/ERO1L interaction with the target Ab and shows how the expression levels of these proteins could affect the qAb of this Ab-producing rCHO cell line. Biotechnol. Bioeng. 2010;107: 337,346. © 2010 Wiley Periodicals, Inc. [source] Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sitesFEBS JOURNAL, Issue 16 2002Anita Fehér The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases. [source] Engineering of Pichia pastoris for improved production of antibody fragmentsBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Brigitte Gasser Abstract The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor , (MF, leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion. © 2006 Wiley Periodicals, Inc. [source] | ||||||||||