Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli James Samuelson Gene Expression Division This work was supported by NIH-SBIR grant R 43 GM 083413 -01 Lab members: Jianying Luo Julie Choulet Carine Robichon
p 8 CBDek fusion partner for MP expression in E. coli FLAG/ek fusion junction N + - TM 2 p 8 + ++ ++ - + + + periplasm inner membrane cytoplasm CBD = chitin binding domain ek = enterokinase cleavage site New England Biolabs
Hydrophobic character of p 8 CBD is sufficient for SRP recognition Signal peptidase -- + p 8 1. 42 + + + N periplasm 1. 80 + 1. 82 inner membrane cytoplasm + + + CBD Threshold hydrophobicity for SRP recognition is 1. 52 (Dsb. A signal) Hydrophobicity values are from the GES scale New England Biolabs
Membrane targeting by p 8 CBD is SRP(Ffh)-dependent Pho. A N FLAG periplasm TM 2 p 8 Inner membrane CBD Significance: membrane translocation is co-translational New England Biolabs
Are p 8 CBD-membrane protein fusions functional within the E. coli inner membrane?
Functional assay: complementation of a strain grown using conditions to deplete an essential membrane protein Strain JS 7131 [att. B: : Para. BAD-yid. C] transformed with Plasmid Ptac-Tma-yid. C or Ptac-PCBD-Tma-yid. C
Tma. Yid. C-8 HIS 0 -2 periplasm 23 248 337 357 401 407 5 267 316 377 384 425 WT signal cytoplasm +6 +1 N 0 +7 8 HIS C p 8 CBD-Tma. Yid. C-8 HIS N 0 -2 248 337 357 401 407 267 316 377 384 425 p 8 +6 +7 0 8 HIS C CBD New England Biolabs
Comparison of targeting signals Strain: MC 1061 Strain: Signal: WT p 8 CBD M - + IPTG MC 1061 Signal: WT p 8 CBD L - + IPTG (k. Da) 83 80 62 60 50 47. 5 p 8 CBDTma. Yid. C 8 HIS Anti-His tag immunoblot New England Biolabs
NEB Express is superior to MC 1061 Strain: NEB Express Signal: p 8 CBD M - + IPTG Signal: p 8 CBD (k. Da) 47. 5 - + IPTG (k. Da) 83 62 L p 8 CBDTma. Yid. C 8 HIS 80 60 50 p 8 CBDTma. Yid. C 8 HIS Anti-His tag immunoblot NEB Express is a BL 21 derivative New England Biolabs
Yid. C-Oxa 1 p chimera functions in E. coli van Bloois et al. J. Biol. Chem. 280 (2005) 247 43 Mitochondrial Oxa 1 p 0 -2 131 217 247 291 293 148 E. coli Yid. C 201 263 276 310 +1 N +5 +1 +15 C New England Biolabs
Expression of p 8 CBDek-Oxa 8 HIS complements the loss of E. coli Yid. C ek site N Mitochondrial Oxa 1 p 43 131 217 247 291 293 148 201 263 276 310 p 8 CBD 8 x. HIS New England Biolabs
Enterokinase cleavage of p 8 CBDek-Oxa 8 HIS Enterokinase: tetramer 200 140 100 80 60 50 Full-length fusion (monomer) 40 30 20 p 8 CBD anti-p 8 immunoblot New England Biolabs
Improved rbs sequence Ptac rbs. O OXA 1 8 HIS p 8 CBD-OXA 8 HIS p. VIII CBD TM 2 EK Ptac rbs. H p. VIII CBD TM 2 FLAG optional EK site p 8 CBDek-OXA 8 HIS OXA 1 8 HIS polylinker sites Acc 65 I – Eco. RI – Bam. HI – Sal. I – Hind. III – Bsi. WI New England Biolabs
AGGAGGTTTGACCTatg ideal E. coli rbs TGGAAACTTCCTCatg rbs. O (wild-type p 8) AGGACGGCCGGatg rbs. H sequence is from Supp. Fig 2 b of Gardner et. al Nature 403 (2000) New England Biolabs
weaker rbs. H results in higher protein expression at 20 hrs 3 hr expression rbs O rbs H 20 hr expression rbs O rbs H 60 50 40 Oxa 1 p fusion L 1 2 3 4 5 6 7 8 9 10 11 12 Each series = 0, 40 or 400 u. M IPTG NEB Express producing p 8 CBD-Oxa 1 p New England Biolabs
Cells induced to express protein from rbs. H continue to grow NEB Express producing p 8 CBD-Oxa 1 p IPTG = 400 u. M New England Biolabs
weaker rbs. H results in higher protein expression at 20 hrs 3 hr expression rbs O rbs H 20 hr expression rbs O rbs H 60 50 40 Oxa 1 p fusion L 1 2 3 4 5 6 7 8 9 10 11 12 Each series = 0, 40 or 400 u. M IPTG NEB Express producing p 8 CBD-Oxa 1 p New England Biolabs
p 8 CBDek fusion partner: replaces a native targeting signal improves expression resistant to proteolysis multiple detection epitopes multiple affinity tag options (1 FLAG/ek site fusion junction N + - TM 2 p 8 + ++ ++ - + + + periplasm inner membrane cytoplasm CBD New England Biolabs
E. coli expression vector p 8 CBDek Enterokinase site p. T 7 or Ptac p. VIII-CBD MP of interest p 8 CBDek lac. Iq Amp. R or Kan. R New England Biolabs
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