[33] Expression and secretion vectors for yeast

Bitter, Grant A.; Egan, Kevin M.; Koski, Raymond A.; Jones, Matthew O.; Elliott, Steven; Giffin, James C.

doi:10.1016/0076-6879(87)53076-2

Cited by 44 publications

(6 citation statements)

References 46 publications

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“…BiP Overexpression Does Not Significantly Affect Heterologous Secretion-Because copper toxicity effects preclude BiP expression from the CUP1 promoter greater than 2-3-fold above wild-type levels (Figs. 1-4), we constructed an expression plasmid with the KAR2 gene transcribed by the strong constitutive GAPDH glycolytic promoter (Bitter et al, 1987). This construct drives BiP expression to levels 3-5-fold higher than wild type as measured by quantitative Western blots (data not shown).…”

Section: Construction Of a Yeast Strain With Variable Bip Levels-mentioning

confidence: 99%

Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Robinson

Bockhaus

Voegler

et al. 1996

Journal of Biological Chemistry

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Increased levels of the endoplasmic reticulum-resident protein folding chaperone BiP would be expected to either increase protein secretory capacity by improved solubilization of folding precursors or decrease secretory capacity by binding and retaining misfolded proteins. To address this question, the relationship between BiP levels and heterologous secretion in yeast was determined. A yeast strain was constructed in which BiP expression is tunable from 5 to 250% of wild-type levels, and this strain was used to explore the effect of varying BiP level on overall secretion of three heterologous proteins: human granulocyte colony-stimulating factor, Schizosaccharomyces pombe acid phosphatase, and bovine pancreatic trypsin inhibitor. For all three proteins examined, reduction in BiP expression below wild-type level diminished overall secretion, whereas 5-fold BiP overexpression from a constitutive glycolytic promoter did not substantially increase or decrease secretion titers. These results are consistent with a positive role for BiP in promoting membrane translocation and solubilization of folding precursors but are inconsistent with a negative role in proofreading and improper retention of heterologous secreted proteins.

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Section: Construction Of a Yeast Strain With Variable Bip Levels-mentioning

confidence: 99%

Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Robinson

Bockhaus

Voegler

et al. 1996

Journal of Biological Chemistry

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“…Pathway optimization often includes balancing of the expression levels of each pathway enzyme, some of which need to be overexpressed [ 13 ]. It has been shown that higher copy number of genes causes higher level of transcription and therefore more efficient production of (heterologous) proteins [ 14 ]. However, using high copy plasmids such as 2μ-based vectors is not the preferred option, since these plasmids are not mitotically stable and thereby lead to heterogeneous gene expression in a population of cells [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

et al. 2016

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Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

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“…(B) Total RNA was isolated from transformed yeast strains(27) and subjected to primer extension (28) with a 20nucleotide primer (5-TCACCAGTGAGACGGGgAAC-3') complementary to lacZ sense strand sequences 17 bp downstream of the Bam HI site(29). Extension products from 25 d.g of RNA were fractionated on a sequencing gel adjacent to 32P-labeled Hae III fragments from pBR322; the 67-and 110-nudeotide fragments are noted.…”

mentioning

confidence: 99%

Mammalian Glucocorticoid Receptor Derivatives Enhance Transcription in Yeast

Schena

1988

Science

313

183

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In mammalian cells, the glucocorticoid receptor binds specifically to glucocorticoid response element (GRE) DNA sequences and enhances transcription from linked promoters. It is shown here that derivatives of the glucocorticoid receptor also enhance transcription when expressed in yeast. Receptor-mediated enhancement in yeast was observed in fusions of GRE sequences to the yeast cytochrome c1 (CYC1) promoter; the CYC1 upstream activator sequences were not essential, since enhancement was observed in fusions of GREs to mutant CYC1 promoters retaining only the TATA region and transcription startpoints. It is concluded that the receptor operates by a common, highly conserved mechanism in yeast and mammalian cells.

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[33] Expression and secretion vectors for yeast

Cited by 44 publications

References 46 publications

Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

Mammalian Glucocorticoid Receptor Derivatives Enhance Transcription in Yeast

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