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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 R43 GM Lab members: Jianying Luo Julie Choulet Carine Robichon
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for MP expression in E.coli
p8CBDek fusion partner for MP expression in E.coli FLAG/ek fusion junction N - - - + - - periplasm inner membrane p8 TM2 + + + cytoplasm + + - + The p8CBD N-terminal fusion partner was designed to replace the native targeting signal of the MP of interest. This fusion partner is comprised of the M13 bacteriophage coat protein p8 linked to a second TM segment to extend the C-terminus into the periplasm. TM2 was borrowed from an E.coli membrane protein where this segment is known to act as an efficient export signal. The presence of a cytoplasmic loop allows for incorporation of an affinity domain for protein purification. Shown here is the chitin binding domain and we have also incorporated a poly-His sequence at the same location without affecting membrane assembly of the fusion partner. 3 epitopes… + - + CBD CBD = chitin binding domain ek = enterokinase cleavage site New England Biolabs
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p8 Hydrophobic character of p8CBD is sufficient for SRP recognition
Signal peptidase - + - - - periplasm p8 inner membrane 1.42 1.80 1.82 + + cytoplasm + + + + N + CBD The p8CBD fusion partner was designed to utilize the SRP pathway. Recognition by the Signal recognition particle is correlated to the hydrophobicity of the N-terminal signal. In the case, the N-terminal signal peptide is only moderately hydrophobic. And when expressed by itself, p8 inserts into the inner membrane efficiently without the need for SRP. However, de Gier’s group showed that when p8 is fused to a soluble protein, it then travels the SRP pathway. So it is most likely that this segment is being recognized by SRP in p8 fusion proteins. When we extended p8 with hydrophobic TM2 we certainly expected to observe SRP dependence. Huber et al. J. Bac 2005 Threshold hydrophobicity for SRP recognition is 1.52 (DsbA signal) Hydrophobicity values are from the GES scale New England Biolabs
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Membrane targeting by p8CBD is SRP(Ffh)-dependent
PhoA N FLAG periplasm p8 TM2 Inner membrane ffh77 mutation was reported by Tian and Beckwith in The ffh77 allele contains a point mutation resulting in an Ala to Proline substitution at position 37 of Ffh. The Ala to Pro substitution compromises the function of the Ffh protein. We created this mutation in the chromosome of strain MC1061 in order to test the SRP-dependence of the p8CBD fusion partner. Strains encoding this mutation grow at a near-normal rate. However, when an SRP-dependent protein is over-expressed, membrane translocation defects become apparent. Significance: We designed p8CBD to travel the same pathway as endogenous MPs. Explain why… Transition- so we have shown that PhoA can be translocated very efficiently, but what about the over-expression and assembly of MPs?? [next slide] Furthermore are MP fusions functional within the E.coli inner membrane? CBD Significance: membrane translocation is co-translational New England Biolabs
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Are p8CBD-membrane protein fusions functional
within the E.coli inner membrane?
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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
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TmaYidC-8HIS WT signal p8CBD-TmaYidC-8HIS N p8 CBD New England Biolabs
periplasm cytoplasm +7 +6 +1 -2 23 5 267 337 316 377 248 357 401 407 425 384 WT signal 8HIS p8CBD-TmaYidC-8HIS N C +7 +6 -2 267 337 316 377 248 357 401 407 425 384 We evaluated the expression of the YidC family member from Thermatoga maritima. The native protein and the p8CBD fusion protein both complement the loss of E.coli YidC indicating proper membrane assembly. Transition- But most importantly, the p8CBD fusion may be expressed at a higher level in E.coli p8 8HIS CBD New England Biolabs
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Comparison of targeting signals
Strain: MC1061 Strain: MC1061 Signal: WT p8CBD Signal: WT p8CBD M IPTG L IPTG (kDa) (kDa) 83 80 60 p8CBD-TmaYidC8HIS 62 p8CBD-TmaYidC8HIS 50 47.5 Anti-His tag immunoblot New England Biolabs
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NEB Express is superior to MC1061
Strain: NEB Express Strain: NEB Express Signal: p8CBD Signal: p8CBD M IPTG L IPTG (kDa) (kDa) 83 80 62 p8CBD-TmaYidC8HIS 60 p8CBD-TmaYidC8HIS 47.5 50 Anti-His tag immunoblot NEB Express is a BL21 derivative New England Biolabs
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YidC-Oxa1p chimera functions in E. coli
van Bloois et al. J. Biol. Chem. 280 (2005) 247 43 Mitochondrial Oxa1p -2 E.coli YidC 131 217 247 291 293 148 201 263 276 310 +1 Oxa1p is also a member of the YidC family. In yeast, Oxa1p mediates the assembly of respiratory chain complexes within the mitochondrial inner membrane. OXA1 is expressed from a nuclear gene and the protein is targeted to the mitochondrial inner membrane by a matrix targeting peptide (AAs 1-42). In 2005, Joen Luirink’s group showed that a YidC-Oxa1p chimera functions in the place of YidC in E.coli. As the essential function of the protein resides in the C-terminal region, we reasoned that this N-terminal signal anchor could be replaced by the p8CBD fusion partner. N +1 +5 +15 C New England Biolabs YidC-Oxa1p chimera functions in E. coli van Bloois et al. J. Biol. Chem. 280 (2005) 247 43 Oxa1p -2 131 217 247 291 293 YidC 148 201 263 276 310 +1 +1 N +5 +15 C
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Expression of p8CBDek-Oxa8HIS complements the loss of E.coli YidC
ek site Mitochondrial Oxa1p N 43 131 217 247 291 293 p8 148 201 263 276 310 Fusion protein is functional. We demonstrated complementation in JS7131 and then we went on to over-produce the fusion protein in NEB Express. Transition- Native Oxa1p is reported to be a tetramer when purifiied from Neurospora mitochondria. In our study… CBD 8xHIS New England Biolabs
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Enterokinase cleavage of p8CBDek-Oxa8HIS
tetramer 200 140 100 80 Full-length fusion (monomer) 60 50 40 30 We also find that the Oxa1 fusion protein also appears to form a stable tetramer that is persistent during purification and during SDS-PAGE analysis. Also, shown here is a pilot experiment to demonstrate the Oxa1 fusion protein may be cleaved by Enterokinase and detected by the p8 antibody. 20 p8CBD anti-p8 immunoblot New England Biolabs
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Improved rbs sequence
p8CBD-OXA8HIS rbsO pVIII CBD OXA1 8HIS TM2 Ptac optional EK EK site p8CBDek-OXA8HIS rbsH pVIII CBD OXA1 8HIS TM2 FLAG Ptac Induction of the Oxa1 fusion with 100uM IPTG caused the cells to stop growing – as is observed in most cases of MP expression. So we set out to moderate the expression level. But instead of replacing the promoter, we chose to alter the ribosomal binding site. polylinker sites Acc65I – EcoRI – BamHI – SalI – HindIII – BsiWI New England Biolabs
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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) rbsH is reported to be a relatively weak sequence for translation initiation. We confirmed in 2 other experiments that rbsH is, in fact, a weaker Shine Dalgarno sequence than rbsO. However, we find that when rbsH is used to express a heterologous MP, a higher protein expression yield is obtained. New England Biolabs
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weaker rbsH results in higher protein expression at 20 hrs
3 hr expression 20 hr expression rbs O rbs H rbs O rbs H 60 Oxa1p fusion 50 40 L Each series = 0, 40 or 400uM IPTG NEB Express producing p8CBD-Oxa1p New England Biolabs
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Cells induced to express protein from rbsH continue to grow
Remarkably, even after induction with a high level of IPTG we found that the cells continued to grow and produce the fusion protein. Cell density is 2.5-fold higher after 20hrs but… These growth curves correspond to lanes 9 and 12 in the previous slide IPTG = 400uM NEB Express producing p8CBD-Oxa1p New England Biolabs
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weaker rbsH results in higher protein expression at 20 hrs
3 hr expression 20 hr expression rbs O rbs H rbs O rbs H 60 Oxa1p fusion 50 40 L Each series = 0, 40 or 400uM IPTG NEB Express producing p8CBD-Oxa1p New England Biolabs
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resistant to proteolysis multiple detection epitopes
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
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E. coli expression vector p8CBDek
Enterokinase site pT7 or Ptac pVIII-CBD MP of interest p8CBDek Simple vector system 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 Ptac pVIII-CBD MP of interest Ptac pVIII-CBD MP of interest Ptac pVIII-CBD MP of interest pVIII-CBD MP of interest pVIII-CBD MP of interest pVIII-CBD p8CBDek MP of interest pVIII-CBD p8CBDek MP of interest p8CBDek p8CBDek p8CBDek lacIq p8CBDek AmpR lacIq p8CBDek AmpR lacIq AmpR lacIq AmpR lacIq AmpR lacIq AmpR lacIq AmpR
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