http://2008.igem.org/wiki/images/9/9c/JHU_0708_paper_Yeast_vectors_for_the_controlled_expression_of_heterologous_proteins.pdf

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

SUMMARY 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 2~t plasmids of the pRS series [Sikorski and Hieter, Genetics 122 (1989) 19-27; Christianson et al., Gene 110 (1992) 119-122] containing HIS3, TRPI, 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.

Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds (Saccharomyces cerevisiae; plasmid; multicopy vector; polylinker; promoter; heterologous expression; cDNA cloning)

Foreign gene expression in yeast: a review.

https://mmbr.asm.org/content/mmbr/47/1/1.full.pdf

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF

The surface antigen of hepatitis B virus (HBsAg) has been synthesized in the yeast Saccharomyces cerevisiae by using an expression vector that employs the 5′-flanking region of yeast alcohol dehydrogenase I as a promoter to transcribe surface antigen coding sequences. The protein synthesized in yeast is assembled into particles having properties similar to the 22-nm particles secreted by human cells.

Synthesis and assembly of hepatitis B virus surface antigen particles in yeast

A DNA sequence coding for mature human leukocyte interferon D (LeIF-D) was linked with DNA fragments of the 5′-flanking sequences of the Saccharomyces cerevisiae (yeast) alcohol dehydrogenase I gene in a plasmid capable of autonomous replication and selection in both yeast and Escherichia coli. Yeast cells transformed by these plasmids synthesize up to 1 × 1O6 molecules of biologically active LeIF-D per cell.

Expression of a human gene for interferon in yeast.

DNA expression vectors capable, in a transformant strain of yeast, of expressing a polypeptide under the control of a genetically distinct yeast promoter, processes of forming transformant strains of yeast and transformed yeast strains are disclosed

Expression of polypeptides in yeast

Cyclin-dependent kinase (Cdk) mutations that prevent entry into the mitotic cell cycle of budding yeast fail to block meiotic DNA replication, suggesting there may be fundamental differences between these pathways. However, S phase in meiosis was found to depend on the same B-type cyclins (Clb5 and Clb6) as it does in mitosis. Meiosis differs instead in the mechanism that controls removal of the Cdk inhibitor Sic1. Destruction of Sic1 and activation of a Clb5-dependent kinase in meiotic cells required the action of the meiosis-specific protein kinase Ime2, thereby coupling early meiotic gene expression to control of DNA replication for meiosis.

https://science.sciencemag.org/content/sci/281/5384/1854.full.pdf

Regulation of Meiotic S Phase by Ime2 and a Clb5,6-Associated Kinase in Saccharomyces cerevisiae

PROBLEM TO BE SOLVED: To efficiently express an exogenous polypeptide in a yeast by introducing a DNA expression vector capable of expressing an exogenous polypeptide into a yeast under control of a yeast promoter which is genetically different and transforming and culturing the yeast. SOLUTION: A yeast is transformed with an expression vector containing a DNA derived from duplication of bacteria and yeast, a gene for phenotype selection of bacterium and yeast, a structural gene coding for a polypeptide having a biological reactivity and DNA derived from DNA sequence adjacent to an initiation codon of a structural gene of the yeast and capable of functioning in the yeast as a promotor genetically different from the structural gene, properly arranging initiation and termination codons in order to translate the structural gene of a polypeptide having the biological reactivity and carrying out expression of the structural gene under control of the promoter and the transformed yeast is cultured to efficiently produce a polypeptide having biological reactivity which is exogenous to the yeast. COPYRIGHT: (C)1997,JPO

Gustav Ammerer

Papers

Synthesis and assembly of hepatitis B virus surface antigen particles in yeast

Expression of a human gene for interferon in yeast.

Expression of polypeptides in yeast

Regulation of Meiotic S Phase by Ime2 and a Clb5,6-Associated Kinase in Saccharomyces cerevisiae

Expression in yeast