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Co-expression with Duet vectors

Co-expression with Duet vectors

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Co-expression with Duet vectors

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  1. Co-expression with Duet vectors Lab of Molecular Genetics Yong-wook, Choi (2003. 4. 10)

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. Co-expression methods (3/3) Co-expression from different vectors Co-expression from one vector

  8. Dual gene expression vectors • pETDuet-1 & pACYCDuet-1 · Cloning & expression of two target genes · T7lac promoter

  9. 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 His·Tag, 15-a.a S·Tag Lys-Glu-Thr-Ala-Ala-Ala-Lys-Phe-Glu-Arg-Gln-His-Met-Asp-Ser

  10. 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 His·Tag, 15-a.a S·Tag N-terminal C-terminal

  11. Vector & Host strain

  12. 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

  13. Co-expression of two and four target protein * Replicon * pACYCDuet-1 : P15A pETDuet-1 : ColE1 β-gal : β-galactosidase GSTGUS : Glutathione-S-transferase- β-glucuronidase fusion Fluc : Firefly luciferase GFP : Green Fluorescent Protein

  14. 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

  15. The End of Presentation

  16. incompatibility of plasmid(1/2) · Not all types of plasmids in given cell · Two plasmids that can’t 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

  17. 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 Back

  18. pET Vector System