Cloning in Saccharomyces cerevisiae

Cloning in Saccharomyces cerevisiae Background Well known organism – Saccharomyces cerevisiae Genome size : 12 Mbp; Haploid set of chromosomes: 16 Unicellular eukaryote  insight in eukaryotic processes  recombinant protein expression / possibilities of secretion and fermentation  no pyrogenic toxins, but glycosylations are possible  easier to grow than animal cells  cloning of large DNA feasible (YAC vectors)  many auxotrophic and other genetical markers available YEAST vs BACTERIA ADVANTAGE YEAST:  Protein folding  Post translational modifications Secretion (proteins targeted to various organelles or exported for harvesting)  Protein targeting  DNA replication  Cell cycle regulation  Vectors can be maintained as plasmids or integrated YEAST vs Other Eucaryotes ADVANTAGE YEAST:  Less expensive, easier to grow, higher throughput  Shorter Cell Cycle than tissue culture ( Human embryonic kidney cells – day doubling)  Transformation/DNA manipulations easier  Higher protein yield  Protein pharmaceuticals free of human disease  Fewer regulations compared to tissue culture  More extensive genetics YEAST vs Other Eucaryotes DISADVANTAGE YEAST:  Glycosylation in yeast can be different  Proteins may get stuck in ER  It is a lower eukaryote DNA Transformation • DNA transformation is extremely efficient in yeast (>104 transformants/µg DNA possible) • Both linear and circular DNA can be introduced and recombined into the genome • Plasmids capable of self-replication can also be introduced • Several different transformation protocols are available, and include: – Transformation of spheroplasts – Transformation of cells treated with lithium salts – Transformation by electroporation • Mutagenesis can be accomplished in cells transformed with synthetic oligonucleotides (needs a positive selection) Yeast vectors – shuttle vectors Integrative plasmids (YIp) by homologous recombination Episomal plasmids (YEp) carry part of the µ DNA necessary for autonomous replication Autonomously replicating plasmids (YRp) which carry a yeast origin of replication (ARS) Cen plasmids (YCp)carry ARS sequence and a centromeric sequence Integrating Vectors: Plasmids can be inserted into yeast chromosomes Episomal Vectors (eg µ): The µ segment confers the ability to replicate autonomously in the yeast cell Replicating Vectors Yeast DNA containing a centromere is added to the plasmid The nuclear spindle that ensures the proper segregation of chromosomes Artifical chromosomes A plasmid containing a centromere and adding the DNA from yeast telomeres to the end If this construct contains yeast replication origin (autonomous replication sequences, ARS), which behave in many ways like a small yeast chromosome at mitosis Integration into yeast chromosomal DNA by recombination Auxotrophic selection markers • Transform a strain carrying a mutation in the biosynthetic gene, thus the strain is dependent on a growth supplement • Plasmid has the wild-type gene, thus transformation with the plasmid complements the defect on media lacking the supplement URA3 gene: LEU2 gene: HIS3 gene: biosynthesis of uridylate biosynthesis of leucine biosynthesis of histidine Plassmids in bacteria and yeast Eukaryotic activators Gal4 - Gal4 activates transcription of the galactose genes in the yeast S cerevisae - Gal4 binds to four sites (UAS) upstream of GAL1, and activates transcription of GAL1 to 1,000-fold in the presence of galactose Mutation analysis DNA-binding domain of GAL4 (without activation domain) can still bind DNA, but cannot activate transcription Mutation analysis Yeast with a bacterial lacZ reporter plasmid bearing binding sites for bacterial repressor LexA upstream of the GAL1 promoter Modular properties of Transcription activators The Yeast two-hybrid system The Yeast two-hybrid system If the two proteins interact, the reporter gene (here: HIS3) is switched on and the diploids can grow on -His plates If the two proteins don't interact, the reporter gene remains inactive and  the cells can't grow on ­His plates Yeast 2-Hybrid Assay HIS his- leu- trpDBD bait trp lacZ AD fish DB D AD leu reporter his nucleus Measurable product The Yeast two-hybrid system Current Opinion in Biotechnology 2006, 17:387–393 High-through put approaches of the yeast two-hybrid system Problems and limitations Post-translational modification: glycosylation, disulfide bond formation, phosphorylation Bait fusion protein can activate expression of reporter in the absence of activation-domain fusion partner Some fusion proteins are harmful to yeast False-positive clones Yeast Genetics Lab Advantages and disadvantages of yeast 2-H Advantage -Direct identification of DNA sequence of interacting protein -No antibodies requries -Protein purification not necessary -In vivo-protein in native conformation? -Detect low affinity or transient interactions Disadvantage -Gene encoding target protein must be available -Bait and prey must be soluble for nuclear localization -False positives -Stable expression of fusion protein might be a problem -Not approapriate post-translational modifications Yeast Genetics Lab Yeast Dual Two Hybrid Screen Screen the same PREY against two different BAITS Small molecule yeast two-hybrid screening (A) A scaffold bait is constructed by fusing a DNA binding domain (DBD) to a small molecule binding protein such as dihyrofolate reductase (DHFR) Simultaneously, each yeast cell expresses a particular activation domain (AD)-fused prey from a cDNA library (B) A hybrid compound consisting of a small molecule covalently linked to methotrexate is added, which crosses the yeast cell membrane and binds to the DBD-DHFR bait via its methotrexate part In this way, the other part of the small molecule is displayed by the scaffold bait (C) If the AD-prey binds to the small molecule displayed from the scaffold bait, a functional transcription factor is reconstituted via the small moleculeprotein interaction, resulting in activation of the downstream reporter gene Screening for compounds that inhibit a protein-protein interaction URA3 +5-FOA URA3 + 5-FOA (A) An interacting protein pair is expressed as the fusion of DBD-X [a protein of interest X fused to the DNA binding domain (DBD)] and ADY [a protein of interest Y fused to the activation domain (AD)] The reconstitution of an artificial transcription factor in the yeast two-hybrid system activates the downstream reporter gene, which converts a compound added to the medium into a toxic end product, resulting in yeast cell death (B) Addition of a compound, which interferes with the protein-protein interaction, prevents reconstitution of the hybrid transcription factor Consequently, the reporter gene is not activated, and the yeast grows on selective medium Reverse two-hybrid system ... moleculeprotein interaction, resulting in activation of the downstream reporter gene Screening for compounds that inhibit a protein-protein interaction URA3 +5-FOA URA3 + 5-FOA (A) An interacting protein... same PREY against two different BAITS Small molecule yeast two-hybrid screening (A) A scaffold bait is constructed by fusing a DNA binding domain (DBD) to a small molecule binding protein such as... of DNA sequence of interacting protein -No antibodies requries -Protein purification not necessary -In vivo-protein in native conformation? -Detect low affinity or transient interactions Disadvantage