RESUMO
Recent reports of severe haemolytic reactions upon high dose treatment with new generation intravenous immunoglobulins (IVIGs) prompted us to examine the anti-A and anti-B haemagglutinin content of these therapeutics. We compared four different test methods, namely the indirect and direct haemagglutination test as described in the European Pharmacopoiea (Ph. Eur.) and two commercial gelcard systems with the aim to define the most reliable method for a large-scale comparison of different IVIG products. Absolute titres varied when the same samples were analyzed by the four methods, while the relative ranking of six different IVIG preparations representing different manufacturing classes was identical. New generation IVIGs showed 1-2 titre steps higher anti-A titres than the older products. Haemagglutinin titres of all 48 IVIG batches analyzed were within the current Ph. Eur. specification of ≤1:64 when tested by the official pharmacopoeial method. Based on efficiency, reliability and lower costs, the direct gelcard method could be a valid alternative to the official Ph. Eur. method to serve as a limit test. However, due to the highest intermediate precision, the official Ph. Eur. method seems to be most suitable to compare haemagglutinin titres of different IVIG products.
Assuntos
Hemaglutininas/análise , Imunoglobulinas Intravenosas/análise , Humanos , Reprodutibilidade dos TestesRESUMO
We have constructed 2-micron-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extended period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant hosts. The highest hirudin titers were obtained with a delta CUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two-fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1-promoter-controlled expression cassette on the same plasmid. In such a set-up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper-sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.
Assuntos
Marcadores Genéticos/genética , Vetores Genéticos/genética , Metalotioneína/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte , Mapeamento Cromossômico , Cobre/metabolismo , Cobre/farmacologia , Meios de Cultura , Dosagem de Genes , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos , Hirudinas/genética , Hirudinas/metabolismo , Plasmídeos/antagonistas & inibidores , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoAssuntos
Clonagem Molecular , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Proteínas de Transporte , Dissulfetos/metabolismo , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Hirudinas/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Metalotioneína/metabolismo , Dados de Sequência Molecular , Plasmídeos , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologiaRESUMO
Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations.
RESUMO
Using the anticoagulant, hirudin, from the leech Hirudo medicinalis as a secreted reporter protein, the influence of physiological parameters on activity and regulation of the yeast (Saccharomyces cerevisiae) metallothionein (CUP1) promoter was studied. Induction of CUP1-directed hirudin expression from 2 mu-based vectors was possible at any time point during diauxic batch growth, even in cells approaching stationary phase. The highest titers of hirudin were obtained when the CUP1 promoter was activated immediately following inoculation of the cultures. If such a pseudo-constitutive fermentation strategy was adopted, the promoter was superior to an optimized variant (GAPFL) of the strong, constitutive GAPDH promoter. This superiority was primarily due to the relative independence of CUP1 promoter activity of the physiological status of host cells: whilst the maximal strength of the CUP1 and GAPFL promoters was comparable, CUP1-directed hirudin expression was high in all phases of diauxic batch growth, whereas hirudin production from the GAPFL promoter declined in post-diauxic cultures. High activity of the CUP1 promoter was observed on both a fermentable (glucose) and a non-fermentable (ethanol) carbon source. Hirudin expression could be adjusted to different levels by varying the amount of inducer (cupric sulphate) added to cultures. The copper concentrations required for maximal promoter induction had no negative effects on host growth and interfered with neither hirudin secretion nor with the biological activity of the peptide. Overexpression of the transcriptional activator, ACE1, resulted in increased levels of hirudin mRNA. Hirudin titers increased in parallel to mRNA concentrations in cultures grown in the presence of low concentrations of copper. In contrast, at high copper doses, elevated levels of the ACE1 protein resulted in inferior hirudin production. Cells overexpressing ACE1 while harbouring a CUP1-drived hirudin expression cassette showed slow growth and poor plasmid maintenance. It was tested whether this might be the result of a block in the secretory pathway; however, measurements of intracellular hirudin did not support this hypothesis. The data rather indicated that hirudin production was limited by a metabolic constraint downstream of transcription but upstream of the secretory pathway.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Hirudinas/metabolismo , Metalotioneína/genética , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Actinas/genética , Sequência de Bases , Divisão Celular , Genes Sintéticos , Vetores Genéticos/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Hirudinas/genética , Mitose , Dados de Sequência Molecular , Plasmídeos/genética , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/biossíntese , Transformação GenéticaRESUMO
In the yeast Saccharomyces cerevisiae, accumulation of the non-reducing disaccharide trehalose is triggered by various stimuli that activate the heat-schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat-induced and stationary-phase-induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPS1 or TPS2, encoding subunits of the trehalose-6-phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat-shock or upon initiation of the stationary phase and significantly reduced the levels of heat-induced and stationary-phase-induced thermotolerance. Deletion of NTH1, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat-induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T., De Virgilio, C., Hall, M. N., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 187-193], we present evidence that the function of heat-induced trehalose accumulation may be to increase the thermal stability of proteins.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/fisiologia , Trealose/biossíntese , Aclimatação/genética , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Temperatura Alta , Cinética , Substâncias Macromoleculares , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Monoéster Fosfórico Hidrolases/biossíntese , Monoéster Fosfórico Hidrolases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fosfatos Açúcares/biossíntese , Fatores de Tempo , Trealase/biossíntese , Trealase/genética , Trealose/análogos & derivadosRESUMO
In baker's yeast (Saccharomyces cerevisiae), accumulation of the non-reducing disaccharide, trehalose, is triggered by stimuli that activate the heat-shock response. Previously, trehalose levels have been shown to be closely correlated with thermotolerance, suggesting a protective function of this substance. Genetic evidence in support of this view is presented in an accompanying paper [De Virgilio, C., Hottiger, T., Dominguez, J., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 179-186]. In this study, we have examined the effect of trehalose on the thermal stability of proteins, a parameter thought to be a major determinant of thermotolerance. Physiological concentrations of trehalose (up to 0.5 M) were found to efficiently protect enzymes of yeast (glucose-6P-dehydrogenase, phosphoglucose-isomerase) as well as enzymes of non-yeast origin (bovine glutamic dehydrogenase, EcoRI) against heat inactivation in vitro. Trehalose also reduced the heat-induced formation of protein aggregates. The disaccharide proved to be a compatible solute, as even at very high concentrations (up to 1 M) it did not significantly interfere with the activity of test enzymes. Trehalose was at least as good or better a protein stabilizer than any of a number of other compatible solutes (including sugars, polyalcohols and amino acids), while the structurally related trehalose-6P was devoid of any protective effect. Thermoprotection of enzymes by trehalose was evident even in solutions containing high concentrations of yeast protein or substrate. The data indicate that trehalose accumulation may increase the thermotolerance of yeast by enhancing protein stability in intact cells.
Assuntos
Proteínas Fúngicas/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Saccharomyces cerevisiae/fisiologia , Trealose/fisiologia , Aclimatação/fisiologia , Animais , Bovinos , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/metabolismo , Estabilidade Enzimática , Glucose-6-Fosfato Isomerase/química , Glucosefosfato Desidrogenase/química , Glutamato Desidrogenase/química , Glutamato Desidrogenase/metabolismo , Temperatura Alta , Cinética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Termodinâmica , Trealose/metabolismo , Trealose/farmacologiaRESUMO
In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.
Assuntos
Proteínas de Choque Térmico/metabolismo , Saccharomyces cerevisiae/metabolismo , Trealose/metabolismo , Glucose/metabolismo , Temperatura Alta , Trealase/metabolismoRESUMO
Exponentially growing cells of the fission yeast, Schizosaccharomyces pombe, contained virtually no trehalose at 27 degrees C but rapidly accumulated large quantities during heat shock at 40 degrees C. Activities of trehalose-6-phosphate synthase and trehalase also increased upon heat shock. Thermotolerance of the cells, measured as survival at 52 degrees C, increased in parallel to trehalose accumulation and decreased in parallel to the trehalose levels when cells were shifted back to 27 degrees C. Trehalose levels, activities of enzymes of trehalose metabolism and thermotolerance strongly increased upon heat shock even in the presence of cycloheximide, indicating that none of these effects requires protein synthesis. The data support the hypothesis that trehalose acts as a thermoprotectant in Schizosaccharomyces pombe.
Assuntos
Cicloeximida/farmacologia , Glucosiltransferases/biossíntese , Schizosaccharomyces/enzimologia , Trealase/biossíntese , Trealose/metabolismo , Indução Enzimática , Temperatura Alta , Cinética , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Trehalose content and thermotolerance were closely correlated in wild type yeast (Saccharomyces cerevisiae) and in cyr1-2 and bcy1-1 mutants both during exponential growth at 27 degrees C and during heat shock at 40 degrees C. Trehalose levels were high when heat shock proteins (hsps) were expected to be induced and low when hsps were presumably absent. It was tried to uncouple trehalose biosynthesis and hsp-induction. Various non-heat stresses affected trehalose levels of wild type cells in a similar way as they would have affected hsps. However, no trehalose was accumulated when cells were treated with canavanine, a well-known inducer of hsps but not of the thermotolerant state.
Assuntos
Adenilil Ciclases/genética , Proteínas Fúngicas/genética , Proteínas de Choque Térmico/biossíntese , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Trealose/biossíntese , Proteínas ras , Dissacarídeos , Temperatura Alta , Cinética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Heat shock resulted in rapid accumulation of large amounts of trehalose in Saccharomyces cerevisiae. In cultures growing exponentially on glucose, the trehalose content of the cells increased from 0.01 to 1 g/g of protein within 1 h after the incubation temperature was shifted from 27 to 40 degrees C. When the temperature was readjusted to 27 degrees C, the accumulated trehalose was rapidly degraded. In parallel, the activity of the trehalose-phosphate synthase, the key enzyme of trehalose biosynthesis, increased about sixfold during the heat shock and declined to the normal level after readjustment of the temperature. Surprisingly, the activity of neutral trehalase, the key enzyme of trehalose degradation, also increased about threefold during the heat shock and remained almost constant during recovery of the cells at 27 degrees C. In pulse-labeling experiments with [14C]glucose, trehalose was found to be turned over rapidly in heat-shocked cells, indicating that both anabolic and catabolic enzymes of trehalose metabolism were active in vivo. Possible functions of the heat-induced accumulation of trehalose and its rapid turnover in an apparently futile cycle during heat shock are discussed.
Assuntos
Dissacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Trealose/metabolismo , Cromatografia Líquida de Alta Pressão , Glucosiltransferases/metabolismo , Temperatura Alta , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Trealase/metabolismo , Trealose/biossínteseRESUMO
The trehalose content of exponentially growing Saccharomyces cerevisiae cells rapidly increased in response to a temperature shift from 27 to 40 degrees C and decreased again when the temperature was shifted back from 40 to 27 degrees C. These changes were closely correlated with increases and decreases in the thermotolerance and desiccation tolerance of the cells. Our results support the hypothesis that trehalose functions as a protectant against heat and desiccation.