Scale down of recombinant protein production: a comparative study of scaling performance

A large bioreactor is heterogeneous with respect to concentration gradients of substrates fed to the reactor such as oxygen and growth limiting carbon source. Gradient formation will highly depend on the fluid dynamics and mass transfer capacity of the reactor, especially in the area in which the substrate is added. In this study, some production-scale (12 m³ bioreactor) conditions of a recombinant Escherichia coli process were imitated on a laboratory scale. From the large-scale cultivations, it was shown that locally high concentration of the limiting substrate fed to the process, in this case glucose, existed at the level of the feedpoint. The large-scale process was scaled down from: (i) mixing time experiments performed in the large-scale bioreactor in order to identify and describe the oscillating environment and (ii) identification of two distinct glucose concentration zones in the reactor. An important parameter obtained from mixing time experiments was the residence time in the feed zone of about 10 seconds. The size of the feed zone was estimated to 10%. Based on these observations the scale-down reactor with two compartments was designed. It was composed of one stirred tank reactor and an aerated plug flow reactor, in which the effect of oscillating glucose concentration on biomass yield and acetate formation was studied. Results from these experiments indicated that the lower biomass yield and higher acetate formation obtained on a large scale compared to homogeneous small-scale cultivations were not directly caused by the cell response to the glucose oscillation. This was concluded since no acetate was accumulated during scale-down experiments. An explanation for the differences in results between the two reactor scales may be a secondary effect of high glucose concentration resulting in an increased glucose metabolism causing an oxygen consumption rate locally exceeding the transfer rate. The results from pulse response experiments and glucose concentration measurements, at different locations in the reactor, showed a great consistency for the two feeding/pulse positions used in the large-scale bioreactor. Furthermore, measured periodicity from mixing data agrees well with expected circulation times for each impeller volume. Conclusions are drawn concerning the design of the scale-down reactor.     

Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109

Two Escherichia coli strains, widely used for the production of various recombinant proteins, were compared for their pre-induction growth and acetate accumulation patterns. The strains studied were E. coli BL21 (lambdaDE3), transformed with a plasmid encoding Pseudomonas exotoxin A, and an E. coli K12 derived strain, JM109, carrying a plasmid encoding maltose-binding protein fused with HIV protease. Cultures were grown in controlled bench-top fermentors to the optimal pre-induction density in both high glucose batch and low glucose fed batch strategies. The results showed the superiority of E. coli BL21 (lambdaDE3) as a host for a recombinant protein expression system. For example, JM109 responds differently to high glucose concentration and to low glucose concentration. Its acetate concentration was as high as 10 g/L in a batch mode and 5 g/L in a fed batch mode. In comparison, strain BL21 (lambdaDE3) reached 2 g/L acetate when grown in batch mode and not more than 1 g/L acetate when grown in a fed batch mode. E. coli BL21 (lambdaDE3), most likely, possesses an acetate self-control mechanism which makes it possible to grow to the desired pre-induction density in a high glucose medium using simple batch propagation techniques. Such a technique is cost effective, reproducible, and easy to scale up. (c) 1996 John Wiley & Sons, Inc.     

Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

PYCNOCOCCUS PROVASOLII GEN. ET SP. NOV., A COCCOID PRASINOXANTHIN-CONTAINING PHYTOPLANKTER FROM THE WESTERN NORTH ATLANTIC AND GULF OF MEXICO1

The new genus Pycnococcus Guillard is based on several clones from the western North Atlantic and Gulf of Mexico. The type and only described species, Pycnococcus provasolii Guillard, sp. nov., is typified by clone Ω48-23 from the North Atlantic. Cells of Pycnococcus provasolii are solitary, spherical, 1.5–4.0 μm in diameter, have a resistant cell wall lacking sporopollenin, and have the ultrastructural characteristics of green algae. With the light microscope they are scarcely distinguishable from cells of other coccoid planktonic organisms. In pigmentation P. provasolii resembles Micromonas pusilla, Mantoniella squamata, and Mamiella gilva in having chl a, much chl b, Mg 2,4-divinylphaeoporphyrin a₅ monomethyl ester (presumably), and prasinoxanthin as a major xanthophyll. The pyrenoid of P. provasolii has a cytoplasmic channel, which is unique among species closely related to it. Flagellates, occurring rarely in culture, are similar to but distinguishable from known Pedinomonas species by size and shape. Pycnococcus provasolii is referred to the new family Pycnococcaceae Guillard, in the order Mamiellales of the class Micromonadophyceae (Chlorophyta). Clones of Pycnococcus provasolii are oceanic in nutritional characteristics, require only vitamin B₁₂ in culture, and are well adapted to growth under blue or blue-violet light of low intensity.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

BIOCHEMICAL TAXONOMY OF MARINE PHYTOPLANKTON BY ELECTROPHORESIS OF ENZYMES. I. THE CENTRIC DIATOMS THALASSIOSIRA PSEUDONANA AND T. FLUVIATILIS1,2

Diatom systematics depends almost entirely upon structure of the silica shell. It is not known to what extent the taxonomic species, as defined by shell structure, corresponds to the genetic species—i.e., to the reproductively isolated population. As an approach to this problem, we report here a comparison of enzymes by electrophoresis. We have examined the genetic constitution of a number of clones of (presumably) the same species for each of 2 closely related, centric diatom species: Thalassiosira pseudonana Hasle and Heimdal and T. fluviatilis Hustedt. The 4 clones of T. fluviatilis form a distinct group, clearly separated from all the T. pseudonana clones. Within T. pseudonana, 4 estuarine clones and one reef clone form a group that is distinctly different from 4 oceanic clones. A single clone of T. pseudonana from the Continental Slope waters is intermediate between these 2 groups and probably shares genes with both groups, indicating that the 2 T. pseudonana groups are not genetically isolated. We conclude that i) within groups, isolates are closely related even though they originated from different continents; and, ii) T. pseudonana is subdivided into ecological races.     

Comparing two fish sampling standards over time: largely congruent results but with caveats

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Carbon emission along a eutrophication gradient in temperate riverine wetlands: effect of primary productivity and plant community composition

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Breakdown of low-level total petroleum hydrocarbons (TPH) in contaminated soil using grasses and willows

A phytoremediation study targeting low-level total petroleum hydrocarbons (TPH) was conducted using cool- and warm-season grasses and willows (Salix species) grown in pots filled with contaminated sandy soil from the New Haven Rail Yard, CT. Efficiencies of the TPH degradation were assessed in a 90-day experiment using 20–8.7–16.6 N-P-K water-soluble fertilizer and fertilizer with molasses amendments to enhance phytoremediation. Plant biomass, TPH concentrations, and indigenous microbes quantified with colony-forming units (CFU), were assessed at the end of the study. Switchgrass grown with soil amendments produced the highest aboveground biomass. Bacterial CFU's were in orders of magnitude significantly higher in willows with soil amendments compared to vegetated treatments with no amendments. The greatest reduction in TPH occurred in all vegetated treatments with fertilizer (66–75%) and fertilizer/molasses (65–74%), followed sequentially by vegetated treatments without amendments, unvegetated treatments with amendments, and unvegetated treatments with no amendment. Phytoremediation of low-level TPH contamination was most efficient where fertilization was in combination with plant species. The same level of remediation was achievable through the addition of grasses and/or willow combinations without amendment, or by fertilization of sandy soil.