Yeast vectors are useful for expressing eukaryotic proteins due to yeasts' ability to perform post-translational modifications. Common yeast species used include Saccharomyces cerevisiae, Pichia pastoris, and Schizosaccharomyces pombe. Vectors include integrating, episomal, replicating, centromere, and artificial chromosome plasmids. Vectors are introduced into yeast via transformation or electroporation. Expression is controlled by inducible promoters like GAL or CUP1 in S. cerevisiae and AOX1 in P. pastoris.

1. Cloning and Expression systems in
Yeast
Submitted by
Ranjitha H B
P-2082
BTY

2. Why To Use Yeast Vectors?
• Yeast are eukaryotes, contain complex internal cell structures
• Post-translational modifications
• easy to manipulate as E. coli
• absence of pyrogenic toxins
• cell growth is faster, easier
• less expensive than other eukaryotic cells
• Higher expression levels
• a well-defined genetic system

3. • highly versatile DNA transformation system
• yeast-specific origin of replication (ORI) and a means of selection in
yeast cells, in addition to the bacterial ORI and antibiotic selection
markers
• All contain unique target sites for a number of restriction endonucleases
• can all replicate in E. coli, often at high copy number
• The four most widely used markers are His3, Leu2, Trp1, and Ura3.

4. Introducing DNA into fungi
• use of spheroplasts (i.e. wall-less cells) and was first developed for S.
cerevisiae (Hinnen et al., 1978)
• cell wall is removed enzymically & resulting spheroplasts are fused with
ethylene glycol in the presence of DNA and CaCl2
• spheroplasts are then allowed to generate new cell walls in a stabilizing
medium containing 3% agar
• Electroporation provides a simpler & more convenient
• DNA can also be introduced into yeasts & filamentous fungi by conjugation

5. • Types of Yeast plasmid vector
 Yeast Integrating plasmids (YIp)
 yeast episomal plasmids (YEps)
 yeast replicating plasmids (YRps)
 Yeast centromere plasmids (Ycps)
 yeast artificial chromosomes (YACs)

6. Yeast Integrating plasmids (YIp):
• lack an ORI and must be integrated directly
into the host chromosome via homologous
recombination for efficient multiplication

7.  Yeast Episomal plasmids (YEp):
• Beggs (1978)
• recombining an E. coli cloning vector with the naturally
occurring yeast 2 μm plasmid
• 6.3 kb in size
• High copy number of 50–100 per cell

9.  Yeast Replicating plasmids (YRp):
• contain an Autonomously Replicating Sequence (ARS)
derived from the yeast chromosome.
• can replicate independently of the yeast chromosome
• unstable and may be lost during budding

10. Yeast centromere plasmids
• carry an ars, most of the recombinants were unstable in yeast
• plasmid-borne centromere sequences have the same distinctive
chromatin structure that occurs in the centromere region of yeast
chromosomes (Bloom & Carbon 1982)
• Three characteristics
• Mitotically stable in the absence of selective pressure
• Segregate during meiosis in a Mendelian manner
• found at low copy number in the host cell

11. Yeast artificial chromosomes
• All autonomous plasmid vectors described above are
maintained in yeast as circular DNA molecules, even the YCp
vectors, which possess yeast centromeres
• Thus, none of these vectors resembles the normal yeast
chromosomes, which have a linear structure

12. • DNA insert size- 500 kbp
• The ends of all yeast chromosomes, like those of all
other linear eukaryotic chromosomes, have unique
structures that are called telomeres

13.  Yeast autonomously replicating sequence (ARS1)
• Struhl et al. (1979)
• carry sequences that enable E. coli vectors to replicate in yeast cells
• sequences are known as ars autonomously replicating sequences
• An ars is quite different from a centromere
• ars acts as an origin of replication , Centromer is involved in chromosome
segregation
 Yeast telomeres (TEL)
• Telomeres are the specific sequences (5-TGTGGGTGTGGTG-3), present at ends of
chromosomes in multiple copies, necessary for replication & chromosome
maintenance.

14.  Genes for YAC selection in yeast
• URA3, a gene involved in uracil biosynthesis
• TRP1, a gene involved in tryptophan biosynthesis
• Auxotrophic method of selection
 Bacterial replication origin & a bacterial selectable marker
• To propagate the YAC vector in bacterial cells, prior to insertion of
genomic
• DNA, YAC vectors usually contain the ColE1 ori and the ampicillin
• resistance gene for growth and analysis in E. coli.

16. • Three main species of yeast
 Saccharomyces cerevisiae
 Pichia pastoris
 Schizosaccharomyces pombe

17. Saccharomyces cerevisiae
• Baker’s yeast
• Single-celled eukaryote
• Grows rapidly (a doubling time of approximately 90 min)
• Simple, defined media
• Many, but not all, of the post-translation modifications

18. • Strong constitutive promoters
 Promoters of phosphoglycerate kinase (PGK), glyceraldehyde-
3-phosphate dehydrogenase (GPD) and alcohol
dehydrogenase (ADH1)
• Suffer similar problems as constitutive E. coli expression
systems
• Inducible production
 The GAL System
 The CUP1 System
(Cereghino and Cregg, 1999)

19. The GAL System
• Galactose is converted to glucose-6-phosphate by enzymes of
Leloir pathway
• Leloir pathway structural genes (GAL genes) are expressed at
a high level (0.5–1% of total cellular Mrna), galactose as sole
carbon source
• GAL genes promoter- sites for the transcriptional activator
Gal4p
(St John and Davis, 1981)

20. • Glucose as carbon source - less Gal4p
• Raffinose as carbon source - Gal4p is produced, binds to GAL
structure gene promoters, but a repressor, Gal80p, inhibits its
activity
• Gal80p binds to Gal4p, mask its activation domain
• Unable to recruit the transcriptional machinery
• Galactose- inhibitory effect of Gal80p
(Griggs and Johnston, 1991)
(Lue et al., 1987)

22. The CUP1 System
• Copper ions (Cu2+ and Cu+) are essential, toxic at high levels
• S. cerevisiae, copper homeostasis - uptake, distribution and
detoxification mechanisms
• At high concentrations, detoxification is mediated by a copper ion
sensing metalloregulatory transcription factor- Ace1p
• Upon interaction with copper, Ace1p binds DNA upstream of the
CUP1 gene, encodes a metallothionein protein & induces its
transcription
(Eide, 1998)
(Winge, 1998)

23. • Expression vectors harbouring CUP1 promoter- induce target
gene expression in a copper-dependent fashion
• Can be grown on rich carbon sources, such as glucose, to high
cell density & protein production is initiated by the addition of
copper sulphate (0.5 mM final concentration)
• Drawback-
 presence of copper ions in yeast growth media, and indeed in
water supplies
(Mascorrogallardo et al., 1996)

24. Pichia Pastoris
• Methylotrophic yeast,
• Metabolize methanol as carbon source
• Metabolism of methanol- oxidation of methanol to formaldehyde
using molecular oxygen (O2) by the enzyme alcohol oxidase
• Alcohol oxidase has a poor affinity for O2, compensates by
generating large amounts of enzyme
• Promoter regulating the production of alcohol oxidase (AOX1) can
be used to drive heterologous protein expression
(Koutz et al., 1989)
(Tschopp et al., 1987)

25. • P. pastoris cells containing the expression vector, integrated into
the genome as single or multiple copies
• Grow in glycerol (glucose represses AOX1 transcription, even in
presence of methanol) to extremely high cell density
• Induce target protein expression by addition of methanol
• 0.5 to tens of grams of protein per litre of yeast culture
• Expression of gene encoding recombinant hepatitis B surface
antigen- production of more than 1 g of the antigen from 1 L of P.
pastoris cells
(Hardy et al., 2000)

26. • P. pastoris may have an advantage in the glycosylation of
secreted proteins
• Glycoproteins generated in P. pastoris more closely resemble
the glycoprotein structure of those found in higher eukaryotes
(Cregg, Vedvick and Raschke, 1993)

27. Schizosaccharomyces pombe
• Properties similar to those found in higher-eukaryotic
organisms
• Chromosome structure and function, cell-cycle control, RNA
splicing and codon usage, ideal candidate for the production of
eukaryotic proteins (Giga-Hama and Kumagai, 1999)
• Folding properly, which may reduce protein insolubility

28. • Protein production controlled by the expression from the nmt1 (no
message in thiamine) promoter
• Active when the cells are grown in the absence of thiamine,
allowing downstream transcription of genes under its control, while
in the presence of greater than 0.5 μM thiamine, the promoter is
turned off
(Maundrell, 1993)
(Maundrell, 1990)

29. Properties of different yeast vectors

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