Co-expression with Duet vectors

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Co-expression with Duet vectors

• Lab of Molecular Genetics
• Yong-wook, Choi
• (2003. 4. 10)

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Co-expression in E.coli

• Multi-component protein
• Important in many cellular processes
• Elucidation their mechanism of action
• ? cloning, expression reconstitution of the
• complex in a defined system
• E.coli
• Popular system used to generate the protein
• complex ? easy usage high expression

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Why Co-expression ?

• Separated components of a complex
• ? Often, not soluble mostly due to hydrophobic
  
• patches that are exposed to the solvent.

These patches are usually involved in and
protected by the binding to the other
component(s) of the complex.

higher expression level of soluble protein complex
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Applications of Co-expression

• Protein-Protein interaction
• Analysis of complex multimeric assemblies
• Analysis of multi-subunit complexes
• Identification characterization of the inter-
• acting subunits in multi-protein complexes
• Analysis of biochemical pathways

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Co-expression methods (1/2)

• Co-expression from different vectors
• For plasmid stability...
• Different selectable markers
• Different origins of replication
• Often the copy numbers for the vectors will
• not be the same ? Expression levels
• should be cloned into the different vectors
• ? If possible, under the control of
  different promoters
• cf. incompatibility of plasmid

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Co-expression methods (2/3)

• Co-expression from one vector
• The genes are
• cloned into the same vector
• expressed from one or more promoters
• If cloned under the regulation of one
  promoter
• ? the order in which the genes are cloned,
• usually affects the expression levels of
  the
• proteins.

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Co-expression methods (3/3)

Co-expression from different vectors
Co-expression from one vector
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Dual gene expression vectors

• pETDuet-1 pACYCDuet-1
• Cloning expression of two target genes
• T7lac promoter

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pETDuet-1
Coexpression of two target genes Two multiple
cloning sites (MCS) T7 promoter/lac operator
pBR322-derived ColE1 replicon lacI gene, AmpR
copy number 40 copies/cell 6-a.a
HisTag, 15-a.a STag
Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Me
t-Asp-Ser
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(No Transcript)
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pACYCDuet-1
Coexpression of two target genes Two multiple
cloning sites (MCS) T7 promoter/lac operator
p15A replicon lacI gene, ChloramphenicolR
copy number 10 12 copies/cell 6-a.a
HisTag, 15-a.a STag
N-terminal C-terminal
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(No Transcript)
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Vector Host strain
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Incompatibility (P15A, ColE1)

Chang,A. C. Y. and Cohen, S. N. (1978) J.
Bacteriol. 134, 1141-1156 ColE1-derived
pFC012, pML21 P15A-derived pACYC139, pACYC184
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Co-expression of two and four target protein
Replicon pACYCDuet-1 P15A
pETDuet-1 ColE1
ß-gal ß-galactosidase GSTGUS
Glutathione-S-transferase- ß-glucuronidase
fusion Fluc Firefly luciferase
GFP Green Fluorescent Protein
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Summary

• Two target genes for independent transcription
• from T7lac promoters
• Compatible each other ? Enable coexpression
• ? Four proteins in the same cell
• Protein complex analysis
• Protein-Protein interactions
• Enzymatic pathways

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The End of Presentation
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incompatibility of plasmid(1/2)

• Not all types of plasmids in given cell
• Two plasmids that cant coexist in the same
• host cell for long (same incompatibility
  group)
• Incompatibility due to replication control
• plasmids that share replication control will
  only half
• the copy number they usually would
• Incompatibility due to partitioning
• plasmids that share partition mechanisms
  will compete
• for distribution into daughter cells

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incompatibility of plasmid(2/2)

• When two plasmids share elements
• of the same replication machinery
• compete with each other
• unable to coexist without selection
• in bacterial cultures
• ? Same replicon, incompatibility group
• unable to be maintained within the same
  bacterium
• 
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pET Vector System