cloning and expression system in yeast

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

Why To Use Yeast Vectors?
• Yeast are eukaryotes, contain complex internal cell structures
• Post-translational modificati...

• highly versatile DNA transformation system
• yeast-specific origin of replication (ORI) and a means of selection in
yeas...

Introducing DNA into fungi
• use of spheroplasts (i.e. wall-less cells) and was first developed for S.
cerevisiae (Hinnen ...

• Types of Yeast plasmid vector
 Yeast Integrating plasmids (YIp)
 yeast episomal plasmids (YEps)
 yeast replicating pl...

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

 Yeast Episomal plasmids (YEp):
• Beggs (1978)
• recombining an E. coli cloning vector with the naturally
occurring yeast...

 Yeast Replicating plasmids (YRp):
• contain an Autonomously Replicating Sequence (ARS)
derived from the yeast chromosome...

Yeast centromere plasmids
• carry an ars, most of the recombinants were unstable in yeast
• plasmid-borne centromere seque...

Yeast artificial chromosomes
• All autonomous plasmid vectors described above are
maintained in yeast as circular DNA mole...

• DNA insert size- 500 kbp
• The ends of all yeast chromosomes, like those of all
other linear eukaryotic chromosomes, hav...

 Yeast autonomously replicating sequence (ARS1)
• Struhl et al. (1979)
• carry sequences that enable E. coli vectors to r...

 Genes for YAC selection in yeast
• URA3, a gene involved in uracil biosynthesis
• TRP1, a gene involved in tryptophan bi...

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

Saccharomyces cerevisiae
• Baker’s yeast
• Single-celled eukaryote
• Grows rapidly (a doubling time of approximately 90 mi...

• Strong constitutive promoters
 Promoters of phosphoglycerate kinase (PGK), glyceraldehyde-
3-phosphate dehydrogenase (G...

The GAL System
• Galactose is converted to glucose-6-phosphate by enzymes of
Leloir pathway
• Leloir pathway structural ge...

• Glucose as carbon source - less Gal4p
• Raffinose as carbon source - Gal4p is produced, binds to GAL
structure gene prom...

The CUP1 System
• Copper ions (Cu2+ and Cu+) are essential, toxic at high levels
• S. cerevisiae, copper homeostasis - upt...

• Expression vectors harbouring CUP1 promoter- induce target
gene expression in a copper-dependent fashion
• Can be grown ...

Pichia Pastoris
• Methylotrophic yeast,
• Metabolize methanol as carbon source
• Metabolism of methanol- oxidation of meth...

• P. pastoris cells containing the expression vector, integrated into
the genome as single or multiple copies
• Grow in gl...

• P. pastoris may have an advantage in the glycosylation of
secreted proteins
• Glycoproteins generated in P. pastoris mor...

Schizosaccharomyces pombe
• Properties similar to those found in higher-eukaryotic
organisms
• Chromosome structure and fu...

• Protein production controlled by the expression from the nmt1 (no
message in thiamine) promoter
• Active when the cells ...

Properties of different yeast vectors

THANK YOU

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