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Prof. Fahd M. Nasr

Faculty of SciencesLebanese University

Beirut, Lebanon

https://yeastwonderfulworld.wordpress.com/

Biol328 - B3212Molecular

Biotechnology

Lecture 9

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Generalized eukaryotic cloning vector

Techniques• Transformation of yeast cells

–Electroporation–Lithium acetate treatment, etc.

• Transfection of animal cells–Electroporation–Ca phosphate, DEAE-dextran, etc.

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Yeast as an expression system

• Single-celled, model for genetics, etc.• Strong promoters, naturally occurring

plasmid (2mm) vectors• Post-translational modifications• Engineered to secrete recombinant pro• (Generally Recognized As Safe) organism

(GRAS)

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Recombinant proteins in yeast• Vaccines

– Hepatitis B VSA– Malaria protein– HIV-1 env protein

• Diagnostics– Hepatitis CVP– HIV-1 antigens

• Human therapeutics– EGF, FGF

– Insulin– Insulin-like GF– PDGF– Pro-insulin– GMC-SF– a1 antitrypsin– Factor XIIIa– Hirudin– HGF– Human serum albumin

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Cloning in yeast• Cloning and plasmid preparation from yeast is

very ineffective• Cloning in yeast uses E. coli as a plasmid

production system:– Plasmids are constructed in vitro– Plasmids are transformed into E. coli– Confirm constructions– Plasmids are produced in bacteria....– ....and then transformed into yeast

• Yeast-E. coli shuttle vectors

S. cerevisiae vectors• Five main classes of expression

vectors– Integrative YIps– Replicative YRps– Episomal YEps

• Extensive use for the production of proteins

– Centromeric YCps– Yeast artificial chromosomes YACs

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Shuttle vectors

Integrated, low copy, very stable

High copy, 20-40/cell, relatively stable

High copy, not stable

Low copy, relatively stable

Yeast-E. coli shuttle vectors

• Integrative plasmids (YIp):– Backbone of an E. coli vector pBR322,

pUC19, pBLUESCRIPT, etc.– A yeast selection marker such as URA3,

HIS3, TRP1, LEU2, etc.• Lack any replication origin for yeast• Propagated only through integration into the

genome

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YIp5: pBR322 plus the URA3 gene

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Integration of plasmids into the yeast genome

• Integration by homologous recombination YIp5 will integrate into the URA3 locus

• Integration duplication of the target sequence• The duplicated DNA flanks the vector• More than one yeast gene target within one

gene cut DNA is highly recombinogenic• Integrated plasmids are stably propagated• Occasional pop-out by recombination between the

duplicated sequences

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Getting into strategies• YEps are based on 2mm plasmid

– LEU2 vector leu2 cells to grow on minimal medium

• Selection scheme rely on mutant strains– Auxotrophic for amino acids or nucleotides– Grow on minimal medium + specific

nutrient– leu2 host cell needs leucine

S. cerevisiae promoters• Many promoters are available• Inducible and regulatable are preferred

– Efficient transcription of heterologous genes– Galactose inducible responds rapidly– 1000x increase in presence of galactose

• Repressible, constitutive and hybrid promoters

• Max expression TT from the same gene as the promoter

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Promoters on Vectors

What is best for heterologous genes?

• Clone with DNA sq leader peptide release into the external environment

• Secreted protein vs protein purification from cell lysate

• Factor a signal sq is the most common• Synthetic leader increase secretion• Remove all additional aa. after purification

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Integration of DNA• Plasmid-based systems are unstable

for large-scale approaches• Co-integration of a YSM and a

cloned gene into specific chromosome site

• Single gene copy low yield• Insert into repetitive DNA

YIp Vector for Chromosomal Insertion

Linear DNA works Best

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Yeast Expression Vector (example)

2 micron plasmid

2 micron seq:yeast orioriE = bacterial oriAmpr = bacterial selectionLEU2, e.g. = Leu biosynthesisfor yeast selection oriE

Your favorite gene

(YFG)

LEU2

Ampr

GAPD-term

GAPD-prom

GlycerAldehyde-3 Phosphate

Dehydrogenase

YAC Cloning Vectors

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Intracellular proteins in Yeast

• Most S. cerevisiae intracellular expression sys. same basic features

• General process production of Cu/Zn-SOD– Superoxide anion is a by-product– In humans helps to stimulate the

inflammatory response– Too much of this molecule derivatives can

cause cellular damage

Intracellular proteins in Yeast

• Produce superoxide dismutase (Cu/Zn-SOD) scavenges superoxide anions H2O2 H2O and O2 (catalase)

• Clone human Cu/Zn-SOD cDNA in YEp– YSM (LEU2) + 2mm plasmid origin– E. coli ori + Ampr gene– Insert between P and TT of GAPD gene

GlycerAldehyde Phosphate Dehydrogenase– Transform leu2 yeast strain

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Plasmid Yeast Cloning Vector

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Heterologous Protein Secretion by S. cerevisiae

• Gene must encode leader to pass through secretory system– Use leader sq of factor a (prepro-)– Correct disulfide bond formation,

proteolytic cleavage of leader, etc. occur– Leader removed by endoprotease KR

• Over expression of PDI secretory enzyme also helps (up to 10X)

The end