Heterologous expression lecture

Heterologous Expression in
E. coli & Yeast
Tassanee Lerksuthirat
Research Center, Faculty of Medicine,
Ramathibodi Hospital, Mahidol University
MIC421 Applied Microbiology, KMUTT ~ 9 February 2016
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Products using heterologous expression
• Human therapeutic agents
– Human insulin
– Human epidermal growth factor
• Diagnostic
– HIV-antigen
• Vaccine
– HIV-1 envelope protein
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Recombinant DNA technology
• Cohen, et al. (1973)
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Recombinant DNA technology
Stanley N. Cohen PNAS 2013;110:15521-15529
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Workflow for heterologous expression
General consideration
• Choice of host organism
• Cloning and strain development
• Transcription
• Translation and 3D-structure
• Fate of synthesized
• Recovery
• Availability of the resources
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Comparison of expression systems
Parameter E. coli Yeast
Cell growth Rapid Some what slow
Complexity of growth medium Minimum Minimum
Cost of growth medium Low Some what high
Expression level High Low to High
Extracellular expression Periplasm Medium
Manipulation Easy Some what hard
Posttranslational modifications
Protein folding Usually required May required
N-linked glycosylation None High in mannose
O-linked glycosylation No Yes
Phosphorylation No Yes
Acetylation No Yes
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Example research; E. coli
Gaastra, W., et al. (2010), De Cock A.W., et al. (1987)
Vanittanakom N., et al. (2004), Mendoza L., et al. (1993)
Krajaejun T., et al. (2004), Hanson R. R., et al. (2008)
Picture from Dr. Boonmee Sathapatayavongs http://pythium.pavlab.com/subpage3.html
• Elicitin025
– Putative virulence factor
– Antibody production
– Immunological response
– Potentially diagnostic test
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

What is the characteristic of ELI025?
• Elicitin025
– 112 amino acids
– 10 kDa (W/O SP)
– Signal peptide
– Disulfide bonds
– O-, N- glycosylation
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Plasmid or Vector?
• Plasmid
– Extrachromosomal DNA
– Circular double-stranded
DNA
– Origin of replication (ORI)
– Mainly found in bacteria &
occasionally in yeast
• Vector
– Vehicle plasmid for
maintaining cloned gene
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Plasmid or Vector?
• Vector element
– Origin of replication (ORI)
– Promoter
– Multiple cloning site (MSC)
– Selectable marker
– Antibiotic resistance gene
– Primer binding site
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Plasmid or Vector?
• Selectable marker
– Antibiotic resistant
– Amp, Kan
• Promoter
– Strong
– Regulatable
– Prevent energy drain
• Fusion protein
– Purification
– Expression detection
– His-tag, GST-tag
• Signal sequences
– Periplasmic
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Plasmid or Vector?
• ELI025
– pET28b
– T7/lac promoter
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Plasmid or Vector?
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Host?
Strain Description Use Antibiotic Supplier
BL21 (DE3) deficient in lon and
ompT proteases
general purpose
expression host
none Novagen
Rosetta (DE3) lactose permease
(lacY) mutant,
deficient in lon and
ompT proteases;
contains plasmid
encoding argU, argW,
glyT, IleX, leuW, metT,
proL, thrT,
thrU, and tyrU
expression host;
allows expression of
genes encoding
tRNAs for rare
argenine codons
AGA, AGG, and CGA,
glycine codon GGA,
isoleucine codon
AUA, leucine codon
CUA, and proline
codon CCC
Cam Novagen
Origami (DE3) trxB and gor mutant expression host;
disulfide bond
formation in
cytoplasm
Kan Novagen
Rosetta-gami (DE3) Rosetta + Origami (DE3) Cam + Kan Novagen
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Example research; Yeast
• PAN112
– Putative virulence factor
– Antibody production
– Immunological response
– Potentially diagnostic test
– Unsuccessful in E. coli expression
• Characteristic
– Signal peptide
– Contain 8 disulfide bonds
– N-glycosylation
– 34 kDa (W/O SP)
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

• Available commercial system
– Saccharomyces cerevisiae
– Pichia pastoris
• Disadvantages of S.cerevisiae expression system
– Instability of plasmids
– Hyperglycosylation
– May secrete protein trapped in periplasmic space
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

• Expression in P. pastoris
– work under strong inducible
promoter
• AOX1 (alcohol oxidase 1)
• Regulated by methanol
• 30% of total soluble
protein
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

• Expression of in P. pastoris
– Avoid problems of plasmid instability; vector integrated into
the host chromosome
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

• Expression of PAN112 in P. pastoris
pPICZalphaA-PAN112
4474bp
Zeo(R)
alpha-factor signal peptide
c-myc epitope
6xHis
PAN112
alpha-factor primer
5' AOX1 primer
3' AOX1 primer
AOX1 promoter
TEF1 promoter
EM7 promoter
pUC origin
CYC1 transcription terminator
AOX1 transcription terminator
DraI(4310)
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

• Expression of PAN112 in P. pastoris
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Heterologous expression
• Identify the study purpose
• Choice of host organism
– E. coli or Yeast
• Cloning and strain development
– Targeted protein characteristic
– Codon usage
• Post-modificationRecovery
– Purification system
• Availability of the resources
– Purchase
– Collaborate
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

References
• Cohen, S. N., A. C. Chang, H. W. Boyer and R. B. Helling (1973). "Construction of
biologically functional bacterial plasmids in vitro." Proc Natl Acad Sci U S A 70(11):
3240-3244.
• Cohen, S. N. (2013). "DNA cloning: a personal view after 40 years." Proc Natl Acad
Sci U S A 110(39): 15521-15529.
• Fernandez, J. M. and J. P. Hoeffler (1999). Gene expression systems : using nature
for the art of expression, San Diego, Calif. : Academic Press, c1999.
• Glick, B. R., T. L. Delovitch and C. L. Patten (2014). Medical biotechnology.
• Glick, B. R., J. J. Pasternak and C. L. Patten (2010). "Molecular biotechnology :
principles and applications of recombinant DNA.“
• https://www.addgene.org/
• http://www.merckmillipore.com/
• http://www.thermofisher.com/th/en/home.html
T
a
s
s
a
n
e
e
L
e
r
k
s
u
t
h
i
r
a
t

Heterologous expression lecture

More Related Content

What's hot (20)

Similar to Heterologous expression lecture (20)

More from Tassanee Lerksuthirat (20)

Recently uploaded (20)

Heterologous expression lecture