Abstract:
:The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae, only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in combination with an increase in gene dosage. Most vectors are maintained as plasmids, which forces growth of cultures on poor selective media. Expression of the yeast Gcn4 protein is regulated at the translational level and increases strongly under amino acid starvation. Because under these conditions protein synthesis in general ceases, it is conceivable that regulatory elements that control Gcn4 expression could support selective expression of foreign genes. We cloned DNA fragments residing upstream from the GCN4 coding sequence (including the 5' UTR) and ligated them to a cDNA that encodes the human serum albumin (HSA) gene. These GCN4 regulatory elements induced efficient HSA expression at the translational level under amino acid starvation. The GCN4/HSA cassette promoted efficient, inducible expression on either a multicopy or integrative plasmid. The integrated cassette induced a high level of HSA in dense cultures grown on rich media. Thus, the GCN4-based expression system (pGES) provides high protein quantities. pGES is the first expression vector to be induced at the translational level.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Mimran A,Marbach I,Engelberg Ddoi
10.2144/00283rr04subject
Has Abstractpub_date
2000-03-01 00:00:00pages
552-4, 556, 558-60issue
3eissn
0736-6205issn
1940-9818journal_volume
28pub_type
杂志文章相关文献
BIOTECHNIQUES文献大全abstract::A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline inc...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03356st01
更新日期:2003-12-01 00:00:00
abstract::The ZEBRA protein is a transcriptional activator that induces expression of viral lytic genes in cells harboring latent Epstein-Barr virus (EBV). In this report it is shown that a derivative of ZEBRA that cannot activate transcription (Zd) can be used to detect and characterize activation domains. Three expression vec...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97225rr02
更新日期:1997-05-01 00:00:00
abstract::Here we introduce a modified antibody staining method that uses up to 80% less antibody for flow cytometry. We demonstrate this method for the detection of antigens expressed at high, moderate, or low levels in mouse and rat lymphocytes as well as mouse mammary epithelial cells. We obtained reproducibly accurate resul...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/0000113854
更新日期:2012-07-01 00:00:00
abstract::A method allowing the evaluation of the structure and the calculation of the volume of a biofilm, using an optical microscope, is proposed based on the linear relation between the intensity of a pixel in biofilm images grabbed on the x-y plane and the corresponding number of cells in the z direction, which allows the ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112403
更新日期:2007-05-01 00:00:00
abstract::Mutations in the human MYH7 gene, encoding a slow skeletal muscle/β-cardiac myosin heavy chain, cause different types of myopathies. The nematode model Caenorhabditis elegans has frequently been employed to study the molecular and physiological consequences of MYH7 mutations in muscle function by introducing mutations...
journal_title:BioTechniques
pub_type: 信件
doi:10.2144/btn-2020-0012
更新日期:2020-06-01 00:00:00
abstract::Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2020-0112
更新日期:2020-11-01 00:00:00
abstract::Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design o...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114177
更新日期:2014-06-01 00:00:00
abstract::The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibi...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98244rr02
更新日期:1998-04-01 00:00:00
abstract::We previously reported that cervicovaginal lavages (CVL) contain a factor that enhances the replication of human immunodeficiency virus (HIV) by increasing virus transcription in T cells and monocytoid cells. This factor was named the HIV-inducing factor (HIF). To determine the molecular mass of HIF, we adapted a blot...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/00283st05
更新日期:2000-03-01 00:00:00
abstract::An improved method was developed for in situ reverse transcription-polymerase chain reaction (RT-PCR) to detect and localize mRNA in tissue sections. The coverslip mounted-immersion cycled (COSMIC) in situ RT-PCR technique combines the advantages of solution-phase PCR with the tissue immobilization necessary for in si...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98241st02
更新日期:1998-01-01 00:00:00
abstract::Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports r...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1993-03-01 00:00:00
abstract::Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various orig...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04374DD03
更新日期:2004-10-01 00:00:00
abstract::High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinit...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114473
更新日期:2016-11-01 00:00:00
abstract::As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affin...
journal_title:BioTechniques
pub_type:
doi:
更新日期:1998-09-01 00:00:00
abstract::Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor m...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112169
更新日期:2006-05-01 00:00:00
abstract::In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112476
更新日期:2007-06-01 00:00:00
abstract::A technique that can be used to isolate vector/insert junctions from clones in vectors, such as yeast artificial chromosomes and P1s, and to sequence plasmid inserts more rapidly has been developed. A vector primer is combined with single, randomly chosen oligonucleotides in PCRs, to create pools of products. With 12-...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/19962003486
更新日期:1996-03-01 00:00:00
abstract::Changes in cardiac structure that depart from normal have generally been termed "remodeling". Assessment of ventricular remodeling at the cellular level should include measurement of myocyte dimensions. A well-established and reliable method to assess myocyte remodeling uses isolated cells and the Coulter Counter/Chan...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98253pf02
更新日期:1998-09-01 00:00:00
abstract::A method was developed for the detection of bacterial mRNAs using reverse transcriptase followed by the polymerase chain reaction (PCR) and Southern blot analysis. The method involves brief inhibition of protein synthesis with chloramphenicol, followed by reverse transcription, PCR amplification of cDNA and Southern b...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1991-01-01 00:00:00
abstract::Treatment with 2 mM CuSO4 was used to induce a Drosophila melanogaster metallothionein (Mtn) promoter that had been cloned into a recombinant baculovirus. Careful study revealed that the Mtn promoter functioned as an inducible, if somewhat "leaky" promoter within the context of baculovirus-infected cells. In the proce...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97234rr01
更新日期:1997-10-01 00:00:00
abstract::Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, a...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/04361ST04
更新日期:2004-01-01 00:00:00
abstract::Here we demonstrate the use of a mammalian two-hybrid system to study protein-protein interactions. Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the reconstitution of the function of a transcriptional activator. In this system, one protein of interest is expressed as a fusion to the Gal4...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/97222pf02
更新日期:1997-02-01 00:00:00
abstract::The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids th...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02335st03
更新日期:2002-11-01 00:00:00
abstract::Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as star...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/03343rr01
更新日期:2003-03-01 00:00:00
abstract::We propose two methods for characterizing the spatio-temporal behavior of cell populations in culture. The first method, image auto-correlation microscopy (IACM), allows us to characterize the variation in the number of objects as a function of time, thus enabling the quantification of the clustering properties of cel...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000112478
更新日期:2007-07-01 00:00:00
abstract::We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distri...
journal_title:BioTechniques
pub_type: 杂志文章
doi:
更新日期:1990-03-01 00:00:00
abstract::DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo n...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/000114307
更新日期:2015-07-01 00:00:00
abstract::There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization ...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/02325rr02
更新日期:2002-05-01 00:00:00
abstract::New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic e...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/btn-2020-0135
更新日期:2021-01-25 00:00:00
abstract::The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is for...
journal_title:BioTechniques
pub_type: 杂志文章
doi:10.2144/98255rr02
更新日期:1998-11-01 00:00:00