Yeast Cells Allow High-Level Expression and Formation of Polyomavirus-Like Particles

1999 ◽  
Vol 380 (3) ◽  
pp. 381-386 ◽  
Author(s):  
K. Sasnauskas ◽  
O. Buzaite ◽  
F. Vogel ◽  
B. Jandrig ◽  
R. Razanskas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Yeast Cells ◽  
Human Polyomavirus ◽  
Yeast Expression System ◽  
E Coli ◽  
Human Use ◽  
Capsid Protein Vp1 ◽  
High Level

AbstractPolyomavirus-derived virus-like particles (VLPs) have been described as potential carriers for encapsidation of nucleic acids in gene therapy. Although VLPs can be generated inE. colior insect cells, the yeast expression system should be advantageous as it is well established for the biotechnological generation of products for human use, especially because they are free of toxins hazardous for humans. We selected the yeastSaccharomyces cerevisiaefor expression of the major capsid protein VP1 of a non-human polyomavirus, the hamster polyomavirus (HaPV). Two entire HaPV VP1- coding sequences, starting with the authentic and a second upstream ATG, respectively, were subcloned and expressed to high levels inSaccharomyces cerevisiae. The expressed VP1 assembled spontaneously into VLPs with a structure resembling that of the native HaPV capsid. Determination of the subcellular localization revealed a nuclear localization of some particles formed by the N-terminally extended VP1, whereas particles formed by the authentic VP1 were found mainly in the cytoplasmic compartment.

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

scholarly journals Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

2000 ◽  
Vol 44 (11) ◽  
pp. 2985-2990 ◽  
Author(s):  
Hiroshi Kakeya ◽  
Yoshitsugu Miyazaki ◽  
Haruko Miyazaki ◽  
Katherine Nyswaner ◽  
Brian Grimberg ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Candida Albicans ◽  
Amino Acid ◽  
Genetic Analysis ◽  
Expression System ◽  
Single Copy ◽  
Gene Replacement ◽  
Azole Resistance ◽  
High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

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 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 Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance

2020 ◽  
Author(s):  
Jack James ◽  
Benjamin Yarnall ◽  
Andy Koranteng ◽  
Jane Gibson ◽  
Tahmina Rahman ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Expression System ◽  
Cell Mass ◽  
Cell Loss ◽  
Integral Membrane Protein ◽  
Large White ◽  
Over Expression ◽  
E Coli ◽  
Pet System ◽  
High Level

Abstract Background: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts. Results: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9 % cell loss with survival rate of only < 0.03 %. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~50 % of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins.Conclusions: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9 % cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

CRISPR/Cas9 mediated T7 RNA polymerase gene knock-in in E. coli BW25113 makes T7 expression system work efficiently

2021 ◽  
Author(s):  
Changchuan Ye ◽  
Xi Chen ◽  
Mengjie Yang ◽  
Xiangfang Zeng ◽  
Shiyan Qiao
Keyword(s):  
Rna Polymerase ◽  
Mutant Strain ◽  
Aminolevulinic Acid ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacterial Strains ◽  
T7 Promoter ◽  
E Coli ◽  
T7 Expression System ◽  
High Level

Abstract T7 Expression System is a common method of ensuring tight control and high-level induced expression. However, this system can only work in some bacterial strains in which the T7 RNA Polymerase gene resides in the chromosome. In this study, we successfully introduced a chromosomal copy of the T7 RNA Polymerase gene under control of the lacUV5 promoter into Escherichia coli BW25113. The T7 Expression System worked efficiently in this mutant strain named BW25113-T7. We demonstrated that this mutant strain could satisfactorily produce 5-Aminolevulinic Acid via C5 pathway. A final study was designed to enhance the controllability of T7 Expression System in this mutant strain by constructing a T7 Promoter Variants Library. These efforts advanced E. coli BW25113-T7 to be a practical host for future metabolic engineering efforts.

Identification of regulatory regions within the Ty1 transposable element that regulate iso-2-cytochrome c production in the CYC7-H2 yeast mutant

1984 ◽  
Vol 4 (7) ◽  
pp. 1393-1401
Author(s):  
B Errede ◽  
T S Cardillo ◽  
M A Teague ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Transposable Element ◽  
Yeast Cells ◽  
Yeast Mutant ◽  
Yeast Saccharomyces Cerevisiae ◽  
Specific Restriction ◽  
Restriction Endonuclease Cleavage

The CYC7-H2 mutation in the yeast Saccharomyces cerevisiae was caused by insertion of a Ty1 transposable element in front of the iso-2-cytochrome c structural gene, CYC7. The Ty1 insertion places iso-2-cytochrome c production under control of regulatory signals that are normally required for mating functions in yeast cells. We have investigated the regions of the Ty1 insertion that are responsible for the aberrant production of iso-2-cytochrome c in the CYC7-H2 mutant. Five alterations of the CYC7-H2 gene were obtained by specific restriction endonuclease cleavage of the cloned DNA and ligation of appropriate fragments. The CYC7+, CYC7-H2, and modified CYC7-H2 genes were each inserted into the yeast vector YIp5 and used to transform a cytochrome c-deficient yeast strain. Expression and regulation of each allele integrated at the CYC7 locus have been compared in vivo by determination of the amount of iso-2-cytochrome c produced. These results show that distal regions of the Ty1 element are not essential for the CYC7-H2 overproducing phenotype. In contrast, alterations in the vicinity of the proximal Ty1 junction abolish the CYC7-H2 expression and give rise to different phenotypes.

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.

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