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Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli

Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli. James Samuelson Gene Expression Division. Lab members: Jianying Luo Julie Choulet Carine Robichon. This work was supported by NIH-SBIR grant R43 GM 083413-01. p8CBDek fusion partner

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Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli

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  1. Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli James Samuelson Gene Expression Division Lab members: Jianying Luo Julie Choulet Carine Robichon This work was supported by NIH-SBIR grant R43 GM 083413-01

  2. p8CBDek fusion partner for MP expression in E.coli FLAG/ek fusion junction N - - - + periplasm - - inner membrane p8 TM2 + + + cytoplasm + + + - + - + CBD CBD = chitin binding domain ek = enterokinase cleavage site New England Biolabs

  3. Hydrophobic character of p8CBD is sufficient for SRP recognition Signal peptidase + - - - periplasm - inner membrane p8 1.42 1.80 1.82 + + cytoplasm + + + + N + CBD Threshold hydrophobicity for SRP recognition is 1.52 (DsbA signal) Hydrophobicity values are from the GES scale New England Biolabs

  4. Membrane targeting by p8CBD is SRP(Ffh)-dependent PhoA FLAG N periplasm Inner membrane p8 TM2 CBD Significance: membrane translocation is co-translational New England Biolabs

  5. Are p8CBD-membrane protein fusions functional within the E.coli inner membrane?

  6. Functional assay: complementation of a strain grown using conditions to deplete an essential membrane protein Strain JS7131 [attB::ParaBAD-yidC] transformed with Plasmid Ptac-Tma-yidC or Ptac-PCBD-Tma-yidC

  7. TmaYidC-8HIS WT signal 8HIS p8CBD-TmaYidC-8HIS N 0 0 -2 -2 periplasm 357 357 401 401 23 248 248 337 337 407 407 p8 377 377 5 267 267 316 316 384 384 425 425 cytoplasm +6 +6 +1 8HIS 0 0 N +7 +7 C C CBD New England Biolabs

  8. Comparison of targeting signals Strain: MC1061 Strain: MC1061 p8CBD p8CBD WT WT Signal: Signal: M - + - + IPTG L - + - + IPTG (kDa) (kDa) 80 83 p8CBD-TmaYidC8HIS 60 62 p8CBD-TmaYidC8HIS 50 47.5 Anti-His tag immunoblot New England Biolabs

  9. NEB Express is superior to MC1061 Strain: NEB Express Strain: NEB Express p8CBD p8CBD Signal: Signal: M - + IPTG L - + IPTG (kDa) (kDa) 83 80 62 p8CBD-TmaYidC8HIS p8CBD-TmaYidC8HIS 60 50 47.5 Anti-His tag immunoblot NEB Express is a BL21 derivative New England Biolabs

  10. YidC-Oxa1p chimera functions in E. coli van Bloois et al. J. Biol. Chem. 280 (2005) 247 43 Mitochondrial Oxa1p 0 -2 247 217 291 293 131 E.coli YidC 263 276 310 148 201 +1 +1 N +15 +5 C New England Biolabs YidC-Oxa1p chimera functions in E. coli van Bloois et al. J. Biol. Chem. 280 (2005) 247 43 Oxa1p 0 -2 247 217 291 293 131 YidC 263 276 310 148 201 +1 +1 N +15 +5 C

  11. Expression of p8CBDek-Oxa8HIS complements the loss of E.coli YidC ek site Mitochondrial Oxa1p N 43 247 217 291 293 131 p8 263 276 310 148 201 CBD 8xHIS New England Biolabs

  12. Enterokinase cleavage of p8CBDek-Oxa8HIS Enterokinase: tetramer 200 140 100 80 60 50 Full-length fusion (monomer) 40 30 20 p8CBD anti-p8 immunoblot New England Biolabs

  13. Improved rbs sequence p8CBD-OXA8HIS OXA1 8HIS CBD rbsO pVIII TM2 Ptac optional EK EK site p8CBDek-OXA8HIS OXA1 8HIS CBD rbsH pVIII TM2 FLAG Ptac polylinker sites Acc65I – EcoRI – BamHI – SalI – HindIII – BsiWI New England Biolabs

  14. AGGAGGTTTGACCTatg ideal E.coli rbs TGGAAACTTCCTCatg rbsO (wild-type p8) AGGACGGCCGGatg rbsH rbsH sequence is from Supp. Fig 2b of Gardner et.al Nature 403 (2000) New England Biolabs

  15. weaker rbsH results in higher protein expression at 20 hrs 20 hr expression 3 hr expression rbs H rbs H rbs O rbs O 60 Oxa1p fusion 50 40 L 1 2 3 4 5 6 7 8 9 10 11 12 Each series = 0, 40 or 400uM IPTG NEB Express producing p8CBD-Oxa1p New England Biolabs

  16. Cells induced to express protein from rbsH continue to grow IPTG = 400uM NEB Express producing p8CBD-Oxa1p New England Biolabs

  17. weaker rbsH results in higher protein expression at 20 hrs 20 hr expression 3 hr expression rbs H rbs H rbs O rbs O 60 Oxa1p fusion 50 40 L 1 2 3 4 5 6 7 8 9 10 11 12 Each series = 0, 40 or 400uM IPTG NEB Express producing p8CBD-Oxa1p New England Biolabs

  18. p8CBDek fusion partner: replaces a native targeting signal (174 aa) travels SRP pathway improves expression resistant to proteolysis multiple detection epitopes multiple affinity tag options FLAG/ek site fusion junction N - - - + periplasm - - inner membrane p8 TM2 + + + cytoplasm + + + - + - + CBD New England Biolabs

  19. E. coli expression vector p8CBDek Enterokinase site pT7 or Ptac pVIII-CBD MP of interest p8CBDek lacIq AmpR or KanR New England Biolabs E. coli expression vector p8CBDek E. coli expression vector p8CBDek E. coli expression vector p8CBDek E. coli expression vector p8CBDek E. coli expression vector p8CBDek E. coli expression vector p8CBDek E. coli expression vector p8CBDek Enterokinase site Enterokinase site Enterokinase site Enterokinase site Enterokinase site Ptac Enterokinase site Ptac Enterokinase site Ptac Ptac pVIII-CBD MP of interest Ptac pVIII-CBD MP of interest Ptac pVIII-CBD MP of interest Ptac pVIII-CBD MP of interest pVIII-CBD MP of interest p8CBDek pVIII-CBD MP of interest p8CBDek pVIII-CBD MP of interest p8CBDek p8CBDek p8CBDek p8CBDek lacIq AmpR p8CBDek lacIq AmpR lacIq AmpR lacIq AmpR lacIq AmpR lacIq AmpR lacIq AmpR

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