Investigation of Cloning Strategies for A' thaliana GProtein Subuit in Pichia Pastoris

1
Investigation of Cloning Strategies for A.
thaliana G-Protein ? Subuit in Pichia Pastoris

• Burcu Kaplan
• M.Sc. Project
• 2004

2
OUTLINE

• Aim
• Heterotrimeric G proteins
• Expression system
• Methods Results
• Conclusion
• Future outlook

3
Aim

• Cloning Expression of A. thaliana G protein ?
  subunit (G?)
• Produce significant quantities of G?
• biochemical characterization
• structural studies
• Static
• Dynamic

4
Structural data on A. thaliana G?

• Meaningful comparison with mammalian homologs
• Prediction of functional attributions based on
  this comparison.
• Prediction of the nature of interactions within
  the heterotrimer in plants.

5
Heterotrimeric GTP binding proteins

• Mediators that transmit the external signals via
  receptor molecules to effector molecules
• Heterotrimeric complex is made up of three
  subunits labeled as ?-, ?- and ?-.

6
Heterotrimeric GTP Binding Proteins

• G proteins
• membrane bound trimer
• inactive in the GDP-bound, heterotrimeric state
• activated by receptor-catalyzed guanine
  nucleotide exchange
• GTP binding leads to dissociation of G?GTP from
  G?? subunits and activation of downstream
  processes.
• Associated molecules
• a large number of isomer specific receptor,
  effector and regulator molecules GPCRs
  effectors RGSs

7
G-protein coupled signal transduction pathways

• Bohm et al., 1997

8
Mammalian heterotrimeric G proteins

• 23 G?, 6 G?, 12 G?
• Gs, activates adenylyl cyclase
• Gi, inhibits adenylyl cyclase
• Gq, activates phospholipase C
• G12/13, of unknown function

G?
Hamm, 1998
9
G?
Rens-Domiano S et al., 1995
10
GTP vs. GDP
AGTP?S-bound G?t subunit BGDP-bound chimeric
G?t /G?i subunit
Hamm, 1998
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G??
Hamm, 1998
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The heterotrimer in Arabidopsis thaliana

• G? gene, GPA1 (Ma et al., 1990)
• G? gene, AGB1 (Ma, 1994 Weiss et al., 1994)
• 2 G? genes, AGG1 and AGG2 (Mason and Botella,
  2000, 2001 )

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A.thaliana G?

• 1149 bp gene
• 383 a.a 44,482 Da protein
• organs cell types(Ma et al., 1994 Ma, 2001)
• vegetative tissuesleaves and roots
• floral stems
• floral buds and floral meristems
• Localization (Weiss et al., 1997)
• plasma membrane
• endoplasmic reticulum (ER) membrane

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A.thaliana G? function?

• 36 identical to Gi subfamily
• G?z
• GTP binding (Wise et al.,1997 and Iwasaki et al.,
  1997)
• G?z
• GTPase activity(Iwasaki et al., 1997)
• G?z

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G?z

• cell proliferation
• death via its control of potassium channeling

Jones, 2002
16
G? cell proliferation

• gpa1 mutants
• A. thaliana reduced cell division (Ma, 1994)
• Rice dwarfism and small seeds (Fujisawa et al.,
  1999)
• high levels of GPA1 expression reported in
  meristematic tissue (Weiss et al., 1997)

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G? K channels

• guard cell ion-channel stomatal apertures
• ABA
• - inwardly rectifying K channels. (Wang et
  al.,2001)
• phospholipases C D
• ? gpa1 mutant guard cells (Ullah et al., 2002)
• - gpa1 mutant seeds

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A.thaliana G? model
template transducin (Gi)
Sahin, 2002
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GCR1

• G protein coupled receptor, GCR1, in
• A. thaliana (Pandey and Assmann, 2004)
• Ligand?
• C-terminal domain of all known plant G? is nearly
  100 conserved G?/receptor
  interactions
• One G? in A. thaliana genome

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The heterotrimer
AGOT1 (Lambright et al., 1996) Bmodelled A.
thaliana heterotrimer ( Ullah et al., 2003)
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Expression System

• Pichia pastoris

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Pichia pastoris

• Methylotrophic yeast
• AOX
• Promoter
• Ease of genetic manipulation
• Ability to perform post-translational
  modifications of eukaryotic proteins
• Allows large-scale production in fermentation
  systems

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S. cerevisiae

• Mitotic instability of recombinant strains
• Great extent of undesirable overglycosylation
• Difficulties in adapting expression to
  fermentation

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E. coli

• Premature termination of the translated protein
  (Wise et al., 1997)
• Toxicity to host (Bakkal, 2003)
• Lack of posttranslational modifications
• (Graziano et al., 1989)
• Inclusion bodies

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P. pastoris

• AOX
• AOX1
• AOX2
• Mut phenotype
• Mut GS115
• MutS KM71H
• Expression vectors
• pPICZC
• pPICZ?B
• Homologous recombination

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Experimental outline

• E. coli
• Subcloning using pCRII-TOPO
• Cloning using expression vectors
• pPICZC
• pPICZ?B
• P. pastoris
• Homologous recombination
• Mut phenotype
• GS115Mut
• Km71HMutS
• Expression

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E. coli
PCIT 857
pCRII-TOPO
GPA1 PrimersGPA1-FP GPA1-RP
GPA1 PrimersGPA1-FP GPA1-RP
pPICZ?B
pPICZC
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GPA1

• GPA1-FP(EcoRI)
• 5-ATA CCA GAATTC GTC ATG GGC TTA C-3
• GPA1-RP (XhoI)
• 5-TAT ATA CTCGAG TAA AAG GCC AGC C-3
• pCRII-TOPO
• pPICZC/EcoRI XhoI

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GPA1

• GPA1-FP (EcoRI)
• 5-GCG TC GAATTC CC ATG GGC TTA CTC TG-3
• GPA1-RP(XbaI)
• 5-GAC GC TCTAGA CC TAA AAG GCC AGC CT-3
• pCRII-TOPO
• pPICZ?B/EcoRI XbaI

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Subcloning

• PCR product
• pCRII-TOPO
• Ligation
• TOP10F transformation
• Kanamycin/blue-white selection
• Initial verification
• Isolation
• Digestion
• Sequencing

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From subcloning to expression....
PCIT 857
pCRII-TOPO
GPA1 PrimersGPA1-FP GPA1-RP
GPA1 PrimersGPA1-FP GPA1-RP
NO
NO
pPICZ?B
pPICZC
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Cloning using expression vectors

• Digestion
• PCR products
• Expression vectors
• Purification Ligation
• Ethanol precipitation
• TOP10F transformation
• Zeocin selection

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pPICZCGPA1

• Initial verification
• Colony PCR
• Isolation digestion
• Sequencing
• Construct

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pPICZ?BGPA1

• Initial verification
• Colony PCR
• Isolation digestion
• Sequencing
• Construct

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Transformation of Pichia pastoris

• Homologous recombination
• Linear vector DNA
• Transformation
• Electroporation
• Lithium chloride transformation
• Zeocin selection
• Methanol selection
• Colony PCR

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EXPRESSION

• KM71H transformants
• Induction
• 100 ml BMGY
• 20 ml BMMY
• Control strains
• GS115 Albumin
• KM71H/pPICZC
• Result

• GS115
• transformants
• Induction
• 25 ml BMGY
• 100 ml BMMY
• Control strains
• GS115/pPICZC/lacZ
• GS115/pPICZC
• Result

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GS115 Expression

• Induction of GPA1 expression
• Induction of GPA1 expression

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CONCLUSION

• First report of cloning and expressing of G?
  using a yeast expression system
• Cloning using expression vectors
• Homologous recombination into yeast genome
• Methanol induction
• Expression does not negatively effect yeast
  growth.
• rGpa1 expression can be detected with antibody
  staining

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FUTURE OUTLOOK

• Purification (ProBond) a.a sequence
  verification
• Biochemical characterization
• GTPase activity
• GTP binding
• Structural characterization
• X-ray crystallization
• X-ray solution scattering NMR
• Expression of G? and G? in P. pastoris system
• Expression of heterotrimer
• Mating
• Co-expression of three subunit

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References

• Bohm A, Gaudet R, Sigler PB, Structural aspects
  of heterotrimeric G-protein signaling Current
  Opinion in Biotechnology 8 (1997) 480-487
• Hamm HE, The many faces of G protein signaling
  J.Biol. Chem. 2732 (1998) 669-672
• Fujisawa Y, Kato T, Ohki S, Ishikawa A, Kitano
  H,Sasaki T , Asahi T, Iwasaki Y, Suppression of
  the heterotrimeric G protein causes abnormal
  morphology, including dwarfism, in rice Proc.
  Natl. Acad. Sci. USA 96(1999) 75757580
• Ma H, Yanofsky MF, Meyerowitz EM, Molecular
  cloning an characterization of GPA1, a G protein
  alpha subunit gene from Arabidopsis thaliana
  PNAS 87-10 (1990) 3821-3825
• Ma H, GTP-binding proteins in plants new
  members of an old family Plant Mol. Biol. 26
  (1994) 1611-1636.

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References

• Mason MG, Botella JR, Completing the
  heterotrimer isolation and characterization of
  an Arabidopsis thaliana G protein ?-subunit cDNA
  PNAS 9726 (2000) 14784-14788.
• Mason MG, Botella JR, Isolation of a novel
  G-protein ?-subunit from Arabidopsis thaliana and
  its interaction with Gß Biochim. Biophys. Acta.
  1520 (2001) 147-153
• Pandey S, Assmann S, The Arabidopsis putative G
  protein-copuled receptor GCR1 interacts with the
  G protein ? subunit GPA1 and regulates abscisic
  acid signaling The Plant Cell 16 (2004)
  1616-1632.
• Ullah H, Chen J, Wang S, Jones AM, Role of a
  Heterotrimeric G Protein in Regulation of
  Arabidopsis Seed Germination Plant Physiology
  129 (2002) 897-907.

42
References

• Wang X-Q, Ullah H, Jones AM, Assmann SM, G
  protein regulation of ion channels and abscisic
  acid signaling in Arabidopsis guard cells
  Science 292 (2001) 2070-2072
• Weiss CA, Garnaat CW, Mukai K, Hu Y, Ma H,
  Isolation of cDNAs encoding guanine
  nucleotide-binding protein ?-subunit homologues
  from maize (ZGB1) and Arabidopsis (AGB1) PNAS 91
  (1994) 9554-9558.
• Weiss CA, White E, Huang H, Ma H, The G protein
  ?-subunit (GP?1) is associated with the ER and
  the plasma membrane in meristematic cells of
  Arabidopsis and cauliflower FEBS Letters 407
  (1997) 361-367
• Wise A, Thomas PG, Carr TH, Murphy GA, Millner
  PA, Expression of the Arabidopsis G-protein
  GP?1purification and characterization of the
  recombinant protein Plant Molecular Biology 33
  (1997) 723-728

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GPA1 expression
119 kDa

• Membrane bound protein
• Processing of signal
• folding
• overglycosylation
• Proteases

44
GPA1 expression
119 kDa
46 kDa
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KM71H expression
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P. pastoris colony PCR
primers FP 5 AOX RP 3 AOX

• pPICZC 324 bp
• pPICZ?B592 bp

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Mut phenotype
GS115 transformants
Mut normal growth on MMH MDH
MutS slow growth on MMH
normal on MDH
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Homologous recombination
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Carrier DNA

• Salmon sperm DNA
• fragmented
• 15 kb-2 kb, mean 7 kb
• denatured

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Linerization
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colony PCR
pPICZ?BGPA1
EcoRI XbaI digestion
PrimersGPA1-FP GPA1-RP
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Colony PCR
pPICZCGPA1
EcoRI XhoI digestion
PrimersGPA1-FP GPA1-RP
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Isolation
Digestion
EcoRI digestion
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Double digestion of expression vectors
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pPICZC

• Homologous sequence
• Transcriptional termination
• Bacterial replication
• Selection
• MCS
• c-myc epitope
• His-tag

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pPICZC MCS
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pPICZ?B

• Homologous sequence
• Transcriptional termination
• Bacterial replication
• Selection
• MCS
• c-myc epitope
• His-tag
• secretion signal sequence

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pPICZ?B MCS
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pPICZC-GPA1
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pPICZ?B-GPA1
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Thanks....

• Prof. Zehra Sayers
• Thesis comitte
• Faculty members and postdocs
• Friends.....

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THANK YOU
Ünal
Özge
Süphan
Yener
Burcu
Çetin
Rezarta
Yigitcan
Elanur
Mehmet
Tolga
Kürsat
Güngör
Doganay
Sedef
Filiz
Kiwi
Ayça
Gülözgür
Karahan
Doktor
Ümit
Özgür B.
Yasemin
Fahriye
B.Ö.
I.E.
feridun
RitchieCandice
D.T.
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