Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

doi:10.1016/0378-1119(95)00037-7

Gene

Volume 156, Issue 1, 14 April 1995, Pages 119-122

https://doi.org/10.1016/0378-1119(95)00037-7 Get rights and content

Abstract

An expression system for Saccharomyces cerevisiae (Sc) has been developed which, depending on the chosen vector, allows the constitutive expression of proteins at different levels over a range of three orders of magnitude and in different genetic backgrounds. The expression system is comprised of cassettes composed of a weak CYC1 promoter, the ADH promoter or the stronger TEF and GPD promoters, flanked by a cloning array and the CYC1 terminator. The multiple cloning array based on pBIISK (Stratagene) provides six to nine unique restriction sites, which facilitates the cloning of genes and allows for the directed cloning of cDNAs by the widely used ZAP system (Stratagene). Expression cassettes were placed into both the centromeric and 2μ plasmids of the pRS series [Sikorski and Hieter, Genetics 122 (1989) 19-27; Christianson et al., Gene 110 (1992) 119-122] containing HIS3, TRP1, LEU2 or URA3 markers. The 32 expression vectors created by this strategy provide a powerful tool for the convenient cloning and the controlled expression of genes or cDNAs in nearly every genetic background of the currently used Sc strains.

References (18)

G.A. Bitter et al.
Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter
Gene
(1984)
T.W. Christianson et al.
Multifunctional yeast high-copy-number shuttle vectors
Gene
(1992)
C.L. Denis et al.
mRNA levels for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease upon growth on nonfermentable carbon source
J. Biol. Chem.
(1983)
L. Guarente et al.
Distinctly regulated tandem upstream activation sites mediate catabolite repression of the CYC1 gene of S. cerevisiae
Cell
(1984)
J. Gyuris et al.
Cdil, a human G1 and S phase protein phosphatase that associates with Cdk2
Cell
(1993)
A.M. Musti et al.
Transcriptional mapping of two genes coding for glyceraldehyde-3-phosphate dehydrogenase isolated by sequence homology with the chicken gene
Gene
(1983)
A.B. Rose et al.
Propagation and expression of cloned genes in yeast
Methods Enzymol.
(1990)
K.S. Zaret et al.
DNA sequence required for efficient transcription termination in yeast
Cell
(1982)
F.M. Ausubel et al.
Current Protocols in Molecular Biology
(1991)

There are more references available in the full text version of this article.

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Short communicationYeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds

Abstract

Gene

Gene

J. Biol. Chem.

Cell

Cell

Gene

Methods Enzymol.

Cell

Current Protocols in Molecular Biology

Short communication
Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds