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H Boze

Publications and source records attributed to H Boze.

17 recordsLinked to original sources
PubMed2004

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone.

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes.

Animals↗
PubMed2001

NMR monitoring of accumulation and folding of 15N-labeled protein overexpressed in Pichia pastoris.

Postgenomic studies have led to an increasing demand for isotope-labeled proteins. We present a method for producing large quantities of truly native (15)N-labeled protein. Based on the secretion capabilities of the yeast Pichia pastoris, the recombinant protein is easily purified in a single step as it is secreted. Control of all nitrogen sources permits very high labeling yields. As a result, accumulation and folding of the recombinant protein can be monitored by heteronuclear NMR without purification. Comparison of sample spectra with the spectrum of the purified recombinant protein allows detection of the secreted protein in the culture and monitoring of its folding, from the start of the induction phase. The detection limit for a (15)N-labeled protein is estimated as 20 microM and corresponds, for a 10-kDa protein, to a load of 40 mg/liter in the fermentor. This concentration is reached by most reported preparations in P. pastoris. Further concentration by ultrafiltration would compensate for lower production. This procedure may be useful in many structural genomics and combinatorial chemistry screening projects where most protein productions meet the requirements for this method.

Antigens, Plant↗
PubMed2001

Optimization of the wheat puroindoline-a production in Pichia pastoris.

AIMS: A recombinant puroindoline-a (rPIN-a) was produced using the methylotrophic yeast Pichia pastoris. METHODS AND RESULTS: In fed-batch culture, the production of rPIN-a decreased after 24 h of methanol induction. Most of the rPIN-a was not soluble in the culture medium remaining bound to the cell walls. Soluble and membrane-bound rPIN-a were quantified by ELISA after Triton X-114 phase partitioning. In order to improve the production of rPIN-a, the influence of pH, specific growth rate and the addition of TX-114 was tested on two independent continuous cultures. The production of rPIN-a was improved when continuous culture was carried out at 29 degrees C under acid conditions (pH 5) with a low dilution rate (D=0.025 h(-1)). The addition of 0.01% TX-114 to the medium inverted the ratio between the secreted and the membrane-bound rPIN-a. CONCLUSION: When a continuous culture was carried out under optimized conditions, the rPIN-a production yield was increased 10-fold to 14 mg l(-1) and 80% of the rPIN-a was soluble. SIGNIFICANCE AND IMPACT OF THE STUDY: This study would be helpful to optimize the expression of other membrane-bound proteins in P. pastoris.

Culture Media↗
PubMed1998

High-level secretion of a wheat lipid transfer protein in Pichia pastoris.

Plant nonspecific lipid transfer proteins are small basic proteins with eight cysteine residues, all engaged in disulfide bonds. The sequence encoding the wheat 9-kDa LTP was cloned into the secretion vector pYAM7SP8 giving rise to pYTdltp4.90. Production in shake-flasks and a fermentor led to the synthesis of two major species of LTP: a larger than expected species of 14 kDa and a species of 10 kDa, close to the expected size of wheat LTP. When production was carried out in a fermentor with regulation of pH, oxygen level, and feed rate of carbon source, the 10-kDa species was the main protein at the end-point of culture. The recombinant wheat LTP (rLTP), secreted at a level of 720 mg/liter into the culture medium, is soluble. The rLTP was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography, with a recovery yield of 36%. However, the molecular mass of rLTP, determined by mass spectrometry, is 9996 Da, while its naturally occurring counterpart has a molecular mass of 9607 Da. This discrepancy in size corresponds to a protein carrying three extra amino acids (DKR) at its N-terminal end, and this was confirmed by sequencing. In vitro lipid transfer activity showed that rLTP behaves in a similar way to the naturally occurring protein. These data indicate that Pichia pastoris is an efficient system for production of large quantities of soluble and biologically active rLTP for structure/function analysis.

Amino Acid Sequence↗
PubMed1997

Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway.

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).

Biomass↗
PubMed1995

Glucose metabolism, enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum.

The physiology of Hanseniaspora uvarum K5 was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0.28 h-1, glucose was completely metabolized in biomass and CO2. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.

Acetates↗
PubMed1994

Physiological approach to heterologous human serum albumin production by Kluyveromyces lactis in chemostat culture.

Production of recombinant human serum albumin (rHSA) controlled by the constitutive promoter phosphoglycerate kinase was studied in Kluyveromyces lactis. It was governed by both cell concentration and glycolytic flow. The triggering of the fermentation metabolism by unfavourable culture conditions (pH, pO2, D) caused a decrease in the synthesis of the heterologous protein. The highest productivity (75 mg l-1 per h) and rHSA concentration (62 mg l-1) were obtained in chemostat culture with a dilution rate of 0.12 h-1 and with 38 g l-1 dry weight.

Humans↗
PubMed1994

Development of high-cell-density fermentation for heterologous interleukin 1 beta production in Kluyveromyces lactis controlled by the PHO5 promoter.

The use of a phosphate organic form taken up by Kluyveromyces lactis removes repression of the PHO5 promoter and releases heterologous interleukin 1 beta synthesis while providing sufficient phosphate for growth. The oxidative metabolism of high-cell-density fed-batch and chemostat cultures was thus maintained under derepressed protein synthesis conditions. Interleukin 1 beta production was then growth-associated, an unusual mode of protein synthesis regulation under the control of the PHO5 promoter.

6-Phytase↗
PubMed1992

Transport and hydrolysis of maltose by Schwanniomyces castellii.

Hydrolysis and transport of maltose into Schwanniomyces castellii was studied under aerobic and anaerobic conditions. Amylase and glucosidase were not synthetized in presence of maltose in anaerobic conditions. Maltose permease was synthesized in anaerobiosis and its functioning is not inhibited. Glucose strongly repressed induction. The half-saturation constant for uptake of maltose was 0.06 mM. The rate of uptake of maltose was decreased by 2,4-dinitrophenol, antimycin or sodium azide. The significance of these results in relation to the Kluyver effect is discussed.

2,4-Dinitrophenol↗
PubMed1992

Production of food and fodder yeasts.

A decade or so ago, there was considerable interest in developing single cell protein production from raw materials. Many factors have influenced the development of fodder yeast technology, notably the biochemistry and physiology of the yeast. It is shown that those considerations have led to the choice of a continuous fermentation technology.

Animal Feed↗
PubMed1991

Characterization of alternative respiratory pathways in the yeast Schwanniomyces castellii by the study of mutants deficient in cytochromes a+a3 and/or b.

After a general review of the proposed mechanisms and physiological roles of the alternative respiratory pathways found in various organisms, the studies are focussed on the amylolytic yeast Schwaniomyces castellii. In addition to the cytochrome chain, the wild type presents two alternative pathways insensitive to antimycin A. One is salicylhydroxamic acid (SHAM)-sensitive and azide-insensitive; the other is SHAM-insensitive and sensitive to high azide concentration. Conditions for mutagenesis and screening are described, which allow isolation of mutants deficient in cytochromes a+a3 and/or b in this yeast previously classified as petite negative. The relative proportions of the alternative respiratory pathways are compared in the wild type and mutant strains following inhibition by SHAM and azide at optimal concentration as determined by iso-inhibition curves. The growth of the cytochrome deficient mutants on citrate, a non-fermentable carbon source, and the ability of the wild type to grow on citrate+antimycin A, after a lag of about 10 h, indicate an involvement of the alternative pathway(s) in energy production. Rotenone sensitivity of respiration and ATP level confirm the presence of a functional phosphorylation site 1. The role of each alternative respiratory pathway in energy production is discussed.

Cyanides↗
PubMed1990

Alternative respiration pathways in Schwanniomyces castellii. I. Isolation and characterization of cytochrome-deficient mutants.

We have isolated and studied cytochromic-deficient mutants of the amylolytic yeast Schwanniomyces castellii in order to study the possible contribution of cytochromes to alternative pathways. Three mutants were found, lacking cytochrome b, a + a3, or b and a + a3. All strains presented two alternative pathways, which were induced in the wild strain when cytochromic respiration was suppressed by growth in the presence of inhibitors, or without copper. If cytochromic respiration was absent, the Yxs yields in aerobiosis were higher than in anaerobiosis. This shows that the alternative pathways play a part in energy conservation. Cytochrome a + a3 did not appear to be directly involved in the alternative pathways.

Culture Media↗
PubMed1990

Alternative respiration pathways in Schwanniomyces castellii. II. Characteristics of oxidation pathways.

By using cytochrome-deficient mutants of Schwanniomyces castellii found previously, we measured the inhibition constants of azide and SHAM-alone or combined-for the different oxidative pathways, in order to determine the more suitable concentrations of inhibitors. This allowed us to measure the real capacity of each pathway. We calculated their affinity for oxygen, and determined that O2 was preferencially reduced by the cytochromic pathway, then by the SHAM-sensitive pathway, and finally by the SHAM+AA-insensitive pathway.

Antifungal Agents↗
PubMed1987

Uptake of galactose and lactose by Kluyveromyces lactis: biochemical characteristics and attempted genetical analysis.

Study of the lactose and galactose transport systems in Kluyveromyces lactis has shown that lactose uptake is by active transport. The transport system is under monogenic control and is inducible. Galactose uptake is also by active transport but the system is controlled by two genes which, in the four strains we studied, are present only in K. lactis CBS 2359. Galactose uptake in the other K. lactis strains is by a simple diffusion process.

Biological Transport↗
PubMed1987

The role of genes LAC1 and LAC2 in the biosynthesis of lactose metabolism enzymes by Kluyveromyces lactis.

By crossing different Kluyveromyces lactis strains, the role of genes LAC1, LAC2 and of gene C were analyzed. These genes are involved in the biosynthesis of enzymes for the metabolism of lactose and galactose. They control the biosynthesis of the lactose and galactose transport, of beta-galactosidase and of the three enzymes of the Leloir pathway. The presence of at least one of the LAC gene is required for the biosynthesis to occur. The gene C seems to code for a negative factor which blocks the expression of the LAC1 and LAC2 genes in the absence of an inducer.

Biological Transport↗