Different constructs for the expression of mammalian gamma-glutamyltransferase cDNAs in Escherichia coli and in Saccharomyces cerevisiae

1991 ◽  
Vol 37 (5) ◽  
pp. 662-666 ◽  
Author(s):  
C Angele ◽  
T Oster ◽  
A Visvikis ◽  
J M Michels ◽  
M Wellman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Clinical Chemistry ◽  
Rat Kidney ◽  
Expression System ◽  
Yeast Cells ◽  
Single Chain ◽  
Hep G2 ◽  
Gamma Glutamyltransferase ◽  
Activity Measurements

Abstract To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.

Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H+ exchangers

2000 ◽  
Vol 351 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Catherine P. DARLEY ◽  
Olivier C. M. VAN WUYTSWINKEL ◽  
Karel VAN DER WOUDE ◽  
Willem H. MAGER ◽  
Albertus H. DE BOER
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Fusion ◽  
Direct Evidence ◽  
Protein Targeting ◽  
Expression System ◽  
Yeast Cells ◽  
Null Mutant ◽  
Heterologous Expression System ◽  
Na Ions ◽  
Convenient Model

Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na+ ions into the vacuole is one mechanism to confer Na+-tolerance on these organisms. In the present study we provide direct evidence that the ArabidopsisthalianaAt-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na+/H+ exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na+ accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na+/H+ exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20–40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na+/H+ antiporters.

scholarly journals Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation.

1985 ◽  
Vol 5 (4) ◽  
pp. 610-618 ◽  
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Genetic Recombination ◽  
Shuttle Vector ◽  
Yeast Cells ◽  
Adenine Methylation ◽  
Dna Adenine Methylase ◽  
Allele Specific ◽  
Dam Methylase

The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.

scholarly journals EmrE, a Small Escherichia coli Multidrug Transporter, Protects Saccharomyces cerevisiae from Toxins by Sequestration in the Vacuole

1999 ◽  
Vol 181 (3) ◽  
pp. 949-956 ◽  
Author(s):  
Rodrigo Yelin ◽  
Dvir Rotem ◽  
Shimon Schuldiner
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Methyl Viologen ◽  
Fluorescent Protein ◽  
Copper Ions ◽  
Yeast Cells ◽  
Multidrug Transporter ◽  
Uptake System ◽  
Bacterial Cells

ABSTRACT In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from Escherichia coli, in the yeast Saccharomyces cerevisiae. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up 14C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu2+ ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in 14C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu2+ and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu2+, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.

scholarly journals Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene.

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558 ◽  
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

Application of gene transfer technologies to the production of enzyme reference materials: example of gamma-glutamyltransferase

1993 ◽  
Vol 39 (8) ◽  
pp. 1573-1589 ◽  
Author(s):  
G Siest ◽  
T Oster ◽  
A Visvikis ◽  
C Thioudellet ◽  
C Angèle ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Reference Materials ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Clinical Chemistry ◽  
Expression System ◽  
Experimental Conditions ◽  
Gamma Glutamyltransferase ◽  
Native Proteins ◽  
High Level

Abstract Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.

Excision repair functions in Saccharomyces cerevisiae recognize and repair methylation of adenine by the Escherichia coli dam gene

1986 ◽  
Vol 6 (10) ◽  
pp. 3555-3558
Author(s):  
M F Hoekstra ◽  
R E Malone
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Yeast Cells ◽  
Pyrimidine Dimer ◽  
Repair System ◽  
E Coli

Unlike the DNA of higher eucaryotes, the DNA of Saccharomyces cerevisiae (bakers' yeast) is not methylated. Introduction of the Escherichia coli dam gene into yeast cells results in methylation of the N-6 position of adenine. The UV excision repair system of yeast cells specifically responds to the methylation, suggesting that it is capable of recognizing modifications which do not lead to major helix distortion. The UV repair functions examined in this report are involved in the incision step of pyrimidine dimer repair. These observations may have relevance to the rearrangements and recombination events observed when yeast or higher eucaryotic cells are transformed or transfected with DNA grown in E. coli.

scholarly journals Bacterial Toxin-Antitoxin Gene System as Containment Control in Yeast Cells

2000 ◽  
Vol 66 (12) ◽  
pp. 5524-5526 ◽  
Author(s):  
P. Kristoffersen ◽  
G. B. Jensen ◽  
K. Gerdes ◽  
J. Piškur
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Bacterial Toxin ◽  
Containment Control ◽  
Industrial Fermentation ◽  
Fermentation Processes ◽  
Gene System ◽  
Potential Applications

ABSTRACT The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeastSaccharomyces cerevisiae. Expression of therelE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect ofrelE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.

scholarly journals Studies of Single-Chain Antibody Expression in Quiescent Escherichia coli

2004 ◽  
Vol 70 (5) ◽  
pp. 3005-3012 ◽  
Author(s):  
K. J. Mukherjee ◽  
D. C. D. Rowe ◽  
N. A. Watkins ◽  
D. K. Summers
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Formation Rate ◽  
Antibody Fragment ◽  
Control Culture ◽  
Temperature Sensitive ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Specific Product ◽  
Quiescent Cells

ABSTRACT Quiescent Escherichia coli cells are generated by overexpressing the Rcd transcript in an hns-205 mutant host. The resulting nongrowing, metabolically active cells were used here to express a single-chain antibody fragment (scFv) in shake flask and fermentor cultures. The expression system is based on two plasmids; one carries the product gene expressed from λPL under the control of the cI857 temperature-sensitive repressor, while the second expresses Rcd from λPR. Shifting the culture from 30 to 42°C induces Rcd expression and product expression simultaneously. Our scFv carried a PelB leader, and 90% of the protein was secreted into the culture supernatant. In a batch culture, the supernatant concentration of scFv in the quiescent-cell culture (optical density at 600 nm [OD600] of 3.5) was 37 mg liter−1, compared to a maximum of 13 mg liter−1 in the control culture (final OD600 of 20). In a fed-batch fermentor culture, quiescent cells were held at an OD600 of 20 for 24 h and the extracellular scFv concentration reached a maximum of 150 mg liter−1. A control culture with a similar feed reached an OD600 of 80, but despite the higher density, the extracellular scFv concentration did not exceed 35 mg liter−1. Quiescent cells at an OD600 of 50 exhibited a small decline in the specific product formation rate, but nevertheless, an extracellular scFv concentration of 160 mg liter−1 was achieved in 8 h. The rate of extracellular accumulation was 10-fold greater in the quiescent culture than in the control culture. This study demonstrates that it is possible to establish high-density quiescent E. coli cultures that are capable of efficient synthesis, folding, and export of proteins.

scholarly journals Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

1996 ◽  
Vol 315 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Specific Activity ◽  
Expression System ◽  
Yeast Cells ◽  
Transport Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crude Membrane

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

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