表达TNFR-Fc融合蛋白的GS-CHO细胞动态流加培养过程的设计

人肿瘤坏死因子受体Ⅱ-Fc融合蛋白在治疗风湿性、类风湿性关节炎方面拥有广阔的市场前景和巨大的经济价值。本实验以表达TNFR-Fc融合蛋白的GS-CHO细胞为研究对象,结合细胞生长代谢特性和动力学参数分析,以葡萄糖为关键控制参数,通过测定培养上清的葡萄糖浓度对培养过程中的葡萄糖消耗进行及时的预测,调整流加速率,形成了以满足细胞生长代谢需要为基本原则的动态流加培养过程设计模型。在此控制模型指导下,建立了高效的流加培养过程。使最大活细胞密度和最大融合蛋白浓度分别达9.4×106cells/mL和207mg/L,较批次培养分别提高了3.4倍和3倍。本研究所采用的研究方法和控制策略为优化GS-CHO细胞培养过程和TNFR-Fc融合蛋白成功迈向产业化奠定了基础。     

The loss of antibody productivity in continuous culture of hybridoma cells

Two hybridoma lines, HB8178 and AFP-27, were grown in continuous culture. The concentrations of viable cells as well as those of various nutrients and metabolites reached steady-state values. The concentrations of either total IgG or antigen-specific antibody, however, failed to reach steady-state values but rather continuously decreased over the course of the cultures. The fraction of antibody-producing cells in the total cellular population also continuously decreased in the AFP-27 cultures. Comparison of the specific antibody productivity based on either the entire population or the antibody-producing fraction of the population over time suggests that the decrease in productivity was at least partly due to the occurrence of a nonproducing subpopulation of cells.     

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

Enhanced in vitro translation at reduced temperatures using a cold-shock RNA motif

Cell-free synthesis of recombinant proteins has emerged as an alternative method of protein production although protein yields still cannot compete with in vivo expression techniques. In systems based on S30 extracts of Escherichia coli unfavorable side-reactions are involved in limiting protein yields. Therefore, carrying out cell-free reactions at lower temperatures might be beneficial as side reactions should be decreased. In this study we show that by using the 5′-untranslated region of the cold-shock gene cspA from E. coli as mRNA leader in cell-free reactions, the expression temperature can be decreased and simultaneously leads to an increase in protein yields. A compensation for the lower activity of T7 RNA polymerase at lower temperatures enhances protein synthesis even further. Additionally, this 5′-untranslated region also standardizes the optimal expression temperature of different proteins.     

Degradation of starch in poplar cells at low temperatures

In Vitro Cellular & Developmental Biology - Plant - Carbon is essential for plant growth. Starch, the main storage carbohydrate, plays an important role in the plant life cycle. When poplar...     

Limits to life at low temperatures and at reduced water contents and water activities

Liquid water is generally considered an absolute requisite for functional life; consequently, life is expected to function only over the range of temperatures that permit its existence. These limits, however, do not apply to cell survival. Some cells can survive the closest attainable approach to 0 K, and some can survive the loss of over 99% of their water.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.Operated by Union Carbide Corporation under contract W-7405-eng-26 with the U.S. Department of Energy.     

Cell-free protein synthesis system from Escherichia coli cells cultured at decreased temperatures improves productivity by decreasing DNA template degradation

Cell-free protein synthesis has become one of the standard methods for protein expression. One of the major advantages of this method is that PCR-amplified linear DNA fragments can be directly used as templates for protein synthesis. The productivity of cell-free protein synthesis using linear DNA templates is generally lower than that from plasmid DNA templates, especially when using an Escherichia coli cell extract. In the present study, we found that a simple modification of the protocol for cell extract preparation from E. coli, just by altering the cultivation temperature (37 °C) of the cells to a moderately lower range (20-34 °C), dramatically reduced the linear DNA degradation activity in the cell extract. This modification greatly improved the productivity of cell-free protein synthesis from linear DNA templates. The removal of the RecD protein, one of the components of exonuclease V, from the extract had almost the same effect, indicating that the linear DNA degradation activity in the extract was mainly due to the RecD protein and that its expression level was decreased at the lower cultivation temperature.     

Growth of Thermoanaerobium brockii in batch and continuous culture at supraoptimal temperatures

Summary Thermoanaerobium brockii was grown in batch and continuous culture at supraoptimal temperatures (>65° C). Specific growth rates were lower in batch (_max>1.0 h^-1) than in continuous cultures (_max1.2–1.4 h^-1). Acetone addition to the medium did not increase critical dilution rate significantly. The media used contained significantly less organic material and sulfide than previously reported media; however, yeast extract requirements were shown to be exceptionally high (60% of the glucose concentration used). Organic substrates inhibited growth and product formation in chemostat cultures whereas the slow formation of acetic acid was observed in batch cultures, but also with virtually no growth. The inhibiting concentration was found to be approximately 15 g organic carbon·l^-1. The maintenance requirements of T. brockii were in the same range as expected of aerobic extreme thermophiles (m_s0.5 g·g^-1·h^-1) and could be met only by glucose and not by yeast extract. Maintenance was obviously not independent of specific growth rate. Production of the stereospecific alcohol-aldehyde/ketone oxidore-ductase was strictly growth associated and its formation was not affected by acetone added to medium.     

Increasing productivity in recent european oak trees?

As an attempt to analyze the role of the biosphere in the disturbed carbon cycle, as far as the standing biomass is concerned, a search for growth trends in tree ring series was started. A group of 27 cut oak trees from various locations in Europe and with ages between 25 and 262 years was analyzed in the following way: (1) A two-parameter smoothing function applied to each single tree cancelled out the year-to-year noise of ring widths; (2) this function containing the growth information over the whole life span of a tree was used to compute the (smoothed) width of the 40th and the 80th ring for each tree; (3) each ring width derived from a single tree in this way was plotted vs time, i.e., over the particular year of its formation. A statistical analysis of these plots shows that the variation of ring widths (delta r) in time (t) is best represented by an exponential function of the form delta rn(t) = delta r0n + aebt (n = 40; 80). The pre-industrial value, delta r0n (for t approximately less than 1,800), turns out to be identical with the 2,700 years average of European oak trees, as found by Hollstein (1979) in his dendrochronological work. Several possibilities are discussed what might have caused such growth trend.     

Enhanced productivity of NS0 cells in fed-batch culture with cholesterol nanoparticle supplementation

NS0 cells are an important industrial cell line for the production of therapeutic monoclonal antibodies. Culturing these cells is challenging because they are cholesterol auxotrophs, and providing cholesterol to the cells is hampered by the low solubility of lipids in aqueous medium. Limited loading capacity, precipitation, instability, and toxicity are associated with traditional delivery methods that involve solvents or carrier molecules. In this work, nanoparticle cholesterol mixtures (NCM) were produced by electrohydrodynamic spraying and added directly to a cholesterol auxotroph NS0 cell line. Compared to a cholesterol-cyclodextrin solution and a commercial proprietary cholesterol solution, SyntheChol NS0 supplement, NCM is significantly less cytotoxic. In the fed batch cell culture process, product titer was increased by 32% when the NCM supplement replaced SyntheChol NS0 supplement. An even greater product titer improvement, 64%, was achieved when both NCM and SyntheChol NS0 supplements were used in the fed-batch process.     

Freeze-drying of red blood cells at ultra-Low temperatures

The hemolysis of human red blood cells (RBCs) after freeze-drying and resuspension depends on the vacuum-drying temperature. In an experimental study, RBCs were first solidified based on a modified high-yield cryopreservation protocol in the presence of hydroxyethyl starch and maltose. Afterward, they were vacuum-dried in a special low-temperature freeze-drying device at selected shelf temperatures between -5 and -65 degrees C. Subsequently, the dried samples were resuspended in an isotonic, phosphate-buffered saline solution. The hemolysis was determined according to a modified saline stability test. It decreases with a decreasing shelf temperature until a minimum is reached at -35 degrees C. A further decrease of the shelf temperature has no beneficial effect; the hemolysis even increases. To interpret these results, we assume that the hemolysis depends on two contrary damaging effects: (1) the higher the shelf temperature, the higher the probability of structural damages occurring during drying; (2) the lower the shelf temperature, the lower the driving force for water transport; this may lead to an incomplete intracellular dehydration which means that the cells are not in a glassy state at ambient temperature.