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Yeast Vector System

Yeast Vector System. Laboratory of Molecular & Cellular Proteomics Park Youn Jin. Introduction. yeast episome plasmid, YEp yeast integrative plasmids, YIp yeast centromere plasmid, YCp . YIp Vectors .

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Yeast Vector System

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  1. Yeast Vector System Laboratory of Molecular & Cellular Proteomics Park Youn Jin

  2. Introduction • yeast episome plasmid, YEp • yeast integrative plasmids, YIp • yeast centromere plasmid, YCp

  3. YIp Vectors. • The YIp integrative vectors are vectors that do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector sequence flanked by two direct copies of the yeast sequence. Typically, YIp vectors integrate as a single copy. However, methods to integrate multiple copies and stable cell lines with up to 15-20 copies of recombinant gene integrations have been developed for over-expressing specific genes.YIp plasmids with two yeast segments, such as YFG1 and the URA3 marker, have the potential to integrate at either of the genomic loci, whereas vectors containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of the multiple sites within genome.2

  4. YEp Vectors. • The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 μm plasmid that serves as an origin of replication (2 μm ori). The 2 μm ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. Most YEp plasmids are relatively unstable and even under conditions of selective growth, only 60 to 95 percent of the cells retain the YEp plasmid. The copy number of most YEp plasmids ranges from 10 to 40 copies per cell. Although this system is used for small scale expression studies, the use of YEp vectors in large-scale manufacturing is not advisable.

  5. YCp Vectors. • YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences (CEN), and autonomously replicating sequences (ARS). The YCp vectors are typically present at very low copy numbers from 1 to 3 per cell. These vectors are also relatively unstable and not very useful in high level expression but are used as regular cloning vectors (e.g., pYC2, pBM272).

  6. Selection Markers • Yeast selection markers can be classified into two types: complementation markers and dominant selection markers. Dominant selection markers are antibiotic markers that can be used in yeast such as G418 and cyclohexamide. Complementation markers are marker genes that complement an auxotrophic mutation in the genome like URA3, TRPI, HIS3, and LEU2. These auxotrophic markers are used in selection of recombinants with all three types of expression systems (integration, episomal, and centromeric plasmids).

  7. pYES2 • pYES2 is a 5.9 kb vector designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae. Features of the vectors allow easy cloning of your gene of interest and selection of transformants by uracil prototrophy . • Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose • A versatile multiple cloning site for simplified cloning • CYC1 transcriptional terminator for efficient termination of mRNA • URA3 gene for selection of transformants in yeast host strains with a ura3 genotype • Ampicillin resistance gene for selection in E. coli

  8. THANKS

  9. Promoters • A range of yeast promoters is available for protein expression. Some like ADH2,SUC2 are inducible and others like GAPDH are constitutive in expression. Similar to the selection markers, a wide variety of combinations of promoters, markers, and expression systems are commercially available (Table 2).

  10. pRSXXX series Vector

  11. pRS413 through pRS416 are centromeric plasmids (YCp), and differ from the YIp by the insertion of a centromere (CEN) and autonomously replicating sequence (ARS) • pRS403 through pRS406 are integrative plasmids (YIp) • pRS423 through pRS426 are episomal plasmids (YEp) : and carry instead the origin of replication from the yeast 2μ circle plasmid along with REP3 and FRT sequences • Centromere plasmids contain a yeast centromere (CEN) as well as an autonomously replicating sequence (ARS) like those found on yeast chromosomes.  Centromere plasmids are low copy plasmids, usually 1-4 plasmids per cell.  The centromere makes the plasmid very stable, with a plasmid loss rate of only around 1% per generation under non-selective conditions.  CEN plasmids are thought to act like mini-chromosomes, segregating like chromosomes during mitosis via spindle attachment to the CEN region.

  12. YEps (yeast episomal plasmids) – YEp 13 • shuttle vector • pBR322 + 2um DNA + yeast chromosomal DNA

  13. pBR322 • Size : 4363bp

  14. Yeast 2um plasmid • 6 kb, 70~200 copy number

  15. YIps (yeast intergrative plasmids) • No yeast replication origion

  16. YRps (yeast replication origion ) • Contains yeast replication origion

  17. Yeast Autonomously replicating sequences About 400 origins exist in the 17 chromosomes of S. cerevisiae Each yeast origin sequence, called an autonomously replicating sequence (ARS), confers on a plasmid the ability to replicate in yeast and is a required element in yeast artificial chromosomes Only one essential element is 15-bp segment designated element A. Three other short segments-the B1, B2 and B3 elements-are required for efficient functioning of ARS1 A complex of proteins called ORC(origin-recognition complex) binds specifically to element A in ARS1 in an ATP-dependent manner

  18. 형질 전환 효율YEp > YRp > Yip • Copy numberYEp ≒ YRp > Yip • 형질 전환체 안정성YIp 가 제일 안정

  19. Secretion Signals • A variety of secretion signals are also available for expression in S. cerevisiae. These include: • Prepro alpha factor12 • HSp15012 • PHO112 • SUC212 • KILM1 (killer toxin type 1)12 • GGP113 • These systems and combinations of promoter, vectors, and signal sequence can be used for high-level expression of recombinant proteins in yeast.

  20. CYC1 • Transcription of cloned sequences is controlled by the alternative promoters, ADH2 , PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator • Yeast (1993-12-01)PMID: 8154182 • Yeast cells were transformed by a yeast-E. coli shuttle vector carrying the PHO5 promoter, the p24 gene and the CYC1 transcription terminator. • Gene (1989-07-15)PMID: 2551773 • Then degest pSK43SB-EPO plasmid with EcoR I and Cla I, the EC fragment with an alpha-factor leading sequence, EPO gene and CYC1 [?] terminater were produced. • Chung Kuo I Hsueh Ko Hsueh Yuan Hsueh Pao (1997-10-01) PMID: 10453527

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