Protein Disulfide Isomerase Overexpression
Increases Secretion of Foreign Proteins in
Saccharomyces cerevisiae
Hsuan-Yi Chiang
Jwey-Yuan Chuang
1
Question

What are the two proteins in this case to
examine the effect of overexpression of PDI?
Answer

Human platelet derived growth factor B
homodimer PDGF-BB and
Schizosaccharomyces pombe acid
phosphatase
2
Background

Newly synthesis
peptide were
recognized and
transport into ER.
They are then transport
to Golgi apparatus and
are sorted according
to their final
destination.
3
Yeast expression system


Advantage:
properly folding and assembly
glycosylation and other posttranslational modification.
Disadnavtage:
lower expression level
4
Function of PDI

Protein disulfide
isomerase (E.C. 5.3.4.1)
is an abundant, soluble
protein found in the
lumen of the
endoplasmic reticulum
(ER) of eukaryotic cells.
ELIZABETH A. KERSTEEN1 and RONALD T.
RAINES1,2Catalysis of Protein Folding by Protein
Disulfide Isomerase and Small-Molecule Mimics
ANTIOXIDANTS & REDOX SIGNALING
5: 413-425, 2003
Protein disulfide isomerase

In vitro:
Increase the rate of folding

In vivo in E.coli :
overexpress of PDI homolog will
increase the secretion level

In vivo in eukaryote system?
6
In this case

Although high level transcription of genes is
attainable in yeast systems, secretion of the
protein products is often proportionally
increased and protein are accumulate in ER.
7
Experiment Design
a
a
GADPH
promoter
LEU2
PDI
t
GADPH
promoter
GADPH
promoter
PDGF-BB
TRP
t
BJ5464
YVH10
Acid
Phosphatase
t
TRP
YVH10
PDGF-BB
Acid phosphatase
8
Overexpression of PDI in
S. cerevisiae
BJ5464
acid
phosphatase
BJ5464
PDGF-BB
YVH10
YVH10
BJ5464
BJ5464
YVH10
PDI
GADPH-PDI
Relative PDI level
Human PDGF-BB
S. Pombe
acid phosphatase
YVH10
PDI overexpression increase
secretion of heterologous protein
~10-fold higher
~4-fold higher
A435/mL/min
mg/mL
Secreted human PDGF-BB dimer
Secreted S. pombe
acid phosphatase activity
PDI overexpression reduces the
intracellular accumulation
Material:
 Sec18 mutant strain (CAY41)


sec18 is a protein that mediates vesicular fusion
between vacuolar fragments during mitosis and
between ER and Golgi vesicles during the
transport of proteins
EndoH:
Remove carbohydrate residues from proteins.
11
Fate of a-factor fusion protein
pre
pro
a factor
Target protein
Enter ER
pro
In Golgi
a factor
KEX2 cleavage
a factor
KEX2:
Target protein
A protease that process a-factor in Golgi
Target protein
PDI over expression reduces
intracellular PDGF accumulation
CAY 41
transformed with
control plasmid
BJ5464
YVH10
CAY41
CAY41
Transformed with
PDGF-B vector
Quantitative densitometric
analysis
BJ5464
YVH10
PDI continues to catalyze folding in post ER compartment or accumulated
In KEX2 processed PDGF represents misfolded protein only aggregated in
14
Golgi.
Conclusion & Discussion




PDI overexpression significantly enhanced the
PDGF(10 fold) and Acid phosphatase (4 fold)
secretion.
However, further increase PDI level would decrease
the secretion of target protein.
Overexpression of PDI may relief the pressure of
expressing heterologous protein.
Properly folding of heterologous protein would
increase the efficiency of secretion.


A similar result in yeast with BiP, HSP70 member,
expression.
Different folding requirement of proteins may result
in different secretion enhancement.
15
Criticism

The accuracy of their data?
~10-fold higher
16
Secreted human PDGF-BB dimer
17