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Identification, production and purification

Identification, production and purification. Identification methods Purification strategies Single gene expression and in vitro assembly: Co-expression techniques Technical Annex. Co-expression techniques. Co-expression in E. coli. Co-expression in Insect cells.

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Identification, production and purification

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  1. Identification, production and purification Identification methods Purification strategies Single gene expression and in vitro assembly: Co-expression techniques Technical Annex

  2. Co-expression techniques Co-expression in E. coli Co-expression in Insect cells A case study: the transcription/DNA repair factor TFIIH

  3. Production of multi-protein complexes Expression of subunits separately purifiy and mix mix and purify refold, mix and purify .... Co-transformation of bacteria with (compatible) expr vectors Use of multi-expression vectors Co-infection of insect cells by several viruses Generation of viruses that co-express several genes

  4. Co-purification is a powerfull option F-RAR His-DAB RXR NRs heterodimers- Flag-RAR FL(Insect cells)- His- DAB RXR (E. coli) Expression in Sf9 cells Expression in E. coli Adsorb onto Flag affinity resin IMAC/GF purification wash incubate with excess of purified His-DAB RXR F-RAR His-DAB RXR wash 100 ml Sf9 culture F-RAR 100 ml M2-affinity beads elute with competitor peptide 100 mg His-DAB RXR yield: x mg complex adsorb onto IMAC and elute to remove peptide

  5. Co-expression in E. coli

  6. Ka Ap Ap Cm Strategies • Co-transformation with several expression vectors • different antibiotic resistances • (different origin of replication) • One expression vector carrying several genes under the control of • a single promoter • several promoters (e.g. each one having a single gene

  7. pET vectors T7 promoter ColE1

  8. KpnI SpeI NdeI XhoI NotI BamHI rbs T7 terminator T7 promoter Lac operator MCS Co-transformation and co-expression with pET (or pGEX) vectors T7 expression cassette pET15b pACYC-11b ColE1 Ap p15A Cm pACYC-11b pACYC-11b p15A ori Chloramphenicol resistance

  9. Two vectors co-expression • TFIIE-a pet 28b His-tag • pGEX GST-tag • TFIIE-b pACYC no tag Jawhari A, et alStructure and oligomeric state of human transcription factor TFIIE EMBO Reports 2006 May;7(5):500-5

  10. 94kDa 67kDa 43kDa 30kDa 20kDa 14kDa Dissection of p34-p44 Interaction by Coexpression Aim: find a stable and soluble complex to set up crystallization trials Human p44 Human p34 p44 zinc finger C4 zinc finger C4 zinc finger Full length His Full length 1-252 His 1-242 252-395 His 243-303 252-320 His 1-170 321-395 His 171-303 1-320 24 combinations were tested several times, only 1 gave a positive result CE Bd CE Bd CE Bd CE Bd CE Bd His-p34(1-242) CE: crude extract p44(321-395) Bd: beads

  11. Mapping of residues Involved in p34-p44 Interaction on p44 Surface H376A/H380A D370R/D372R C382A/C385A p44(321-395) His-p34(1-233) D370R D372R D377R C345A C360A C368A E333R 94kDa 67kDa 43kDa 30kDa His-p34(1-233) E333 20.1kDa F331 14kDa p44(321-395) Y346 D372 D377 F344S/Y346S D370 p44(321-395) F374S V366S F344S F374 His-p34(1-233) 94kDa 67kDa Surface representation of p44(321-395) 43kDa 30kDa His-p34(1-233) 20.1kDa 14kDa p44(321-395)

  12. Prom T Gene A pET15b + Prom Gene A Gene B T Prom Gene B Gène B pACYC-11b Prom Prom Gene C T Gene A Gene B Gene D Gene C Gene D E. coli co-expression vectors with compatible replication origins - Concatenation of transcription/translation units: n promoter and n rbs - Concatenation of translation units:1 promoter and n rbs pBR322, p15A and pCDM Amp, Chlo, Spect +

  13. Co-expression of chaperones and rare tRNAs Very few false positives as compared to pull-down, two hybrid and immuno-precipitation experiments. Cheap and adapted to HTS The problems of false negatives

  14. Co-expression in Insect cells

  15. Co-infection for co-expression Sf9 ou H5 Co-infection His-tagged subunit Flag-tagged subunit GST-tagged subunit native subunit I 2 50 mM FT Talon I 2 5mM I 5mM IP Flag Wash MW His-p89 p80 His-tag p62 p44, p52 p34F Flag 1 litre, 300 ml dépot 20 ml

  16. Homologous recombination in insect cells (FlashBac, Pharmingen, tri-ex) Homologous Recombinaison in E.Coli (Bacmids)

  17. Multi gene baculovirus transfer vectors co-expression vectors pAcAB3 derivatives pFastBac Dual derivatives • - Recombination in E. coli or in insect cells • Use of small tags inserted by PCR • orientrations of the promoters (Berger, 2004)

  18. Concatenation of expression units (restriction/ligation) Concatenation of vectors (Cre/Lox in vitro recombination) • Usual NdeI – BamHI sites, or Gateway, for gene cloning whatever the vector. - Choice of the promoter independent of cloning sites. - Choice of the gene ordering. - Possibility to concatenate using either a unique or both promoters.

  19. MultiBac: a new engeniered bacmid • Derived from the DH10Bac bacmid • ChiA gene deleted • - Possibility to integrate genes via a Tn7 and a LoxP site • In the Tn7 entry site: integration of pFastBac derivatives by Tn7 transposition • - In the LoxP site: integration of the transfer vector with the Cre recombinase

  20. Combinatorial applications in protein expression Domain definition Specific modifications

  21. TFIIH: a 10 subunit multi-protein complex Nucleotide Excision Repair Opens DNA around lesion (XPD) Class I and II transcription (basal and activated) Opens DNA at promoter (XPB) Phosphorylates CTD of RNA polymerase II (Cdk7) Inherited dieseases Mutations identifiedin 3/10 subunits Xerderma pigmentosum (XP-B and XP-D) Trichothyodystrophy (TTD-A) Cockayne’ syndrome

  22. Production of the 10 subunit factor……. Functional reconstitution (cf Tirode, 1999 Mol Cell) Production for structural studies: yes, but… expression level and homogeneity are poor dissection of the protein-protein interaction network production of sub-complexes functional analysis

  23. XPB p52 XPB p52 p34 p52 p34 p62 p44 p62 p44 p34 + p62 XPB XPB p44 systematic dissection with insect cells (no or few false negatives) rIIH5 rIIH4 EB EB IP IP XPB XPB p62 * p52,p44 p62 p34 p52 p34(1-233) p44 p34F 1 2 3 4 p34 p62 p44

  24. Pair-wise interactions and deletion analysis XPB H2 C H1 N p62 p8 p52 N C N C p34 N C M cycH p44 N C1 C2 M cdk7 C MAT1 XPD C H1 H2 N Jawhari,A, 2002

  25. Production of a trimeric complex pAcAB3: 1 virus/3 proteins Flag, Strep or His C-terminal on cdk7 Ph + P10 - P10 + • Tag on the right subunit • Improved stochiometry Cdk7 –Flag CyclinH MAT1

  26. Towards a single virus for 10 genes Multi-bac vectors A/B/C/D/E A/B/C A/B A/B/C/D/E A/B/C/D/E^ A/B/C/D/E A/B/C/D/E^ A/B/C A/B/C A/B A/B 2 3 5 5 2 3 5 5 5 genes, 1 virus: co-expressed but non-soluble

  27. Role of p62 PH/PTB domain within TFIIH in transcription and repair FW 1 548 FW 548 109 FW Gervais 2004, Jawhari 2004

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