Rational Design of a Fusion Partner for Membrane Protein Over-Expression in E. coli
This work was supported byNIH-SBIR grantR43 GM 083413-01
James Samuelson
Gene Expression Division
Lab members:Jianying LuoJulie ChouletCarine Robichon
++
++
CBD
fusion junction
cytoplasm
periplasm+----
N
+ +++- -
-
inner membrane
p8 TM2
FLAG/ek
p8CBDek fusion partner for MP expression in E.coli
CBD = chitin binding domain
New England Biolabs
ek = enterokinase cleavage site
Signal peptidase
N
++
+
++
+
--- -
++
CBD
1.42 1.80 1.82
Hydrophobic character of p8CBD is sufficient for SRP recognition
Hydrophobicity values are from the GES scale
p8
Threshold hydrophobicity for SRP recognition is 1.52 (DsbA signal)
cytoplasm
periplasm
inner membrane
New England Biolabs
Membrane targeting by p8CBD is SRP(Ffh)-dependent
MC1061 wt ffh
JL1005 ffh77
PhoA activity
2860
1120
CBD
PhoA
Inner membrane
periplasm
N
p8
FLAG
TM2
Significance: membrane translocation is co-translational
New England Biolabs
Are p8CBD-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 JS7131 [attB::ParaBAD-yidC]
transformed with
Plasmid Ptac-Tma-yidC or Ptac-PCBD-Tma-yidC
N C
periplasm
cytoplasm
+7 0
+6+1
-2 0
23
5 267
337
316 377
248 357 401 407
425384
WT signal
C+7 0
+6
-2 0
267
337
316 377
248 357 401 407
425384
CBD
N
p8CBD-TmaYidC-8HIS
TmaYidC-8HIS
p8
8HIS
8HIS
New England Biolabs
M - + - + IPTG
WT p8CBD
(kDa)
83
62
47.5
Signal:
p8CBD-TmaYidC8HIS
Strain: MC1061
L - + - + IPTG
WT p8CBDSignal:
Strain: MC1061
p8CBD-TmaYidC8HIS
(kDa)
80
6050
Comparison of targeting signals
Anti-His tag immunoblot
New England Biolabs
M - + IPTG
p8CBDSignal:
Strain: NEB Express
p8CBDSignal:
Strain: NEB Express
L - + IPTG
p8CBD-TmaYidC8HIS
p8CBD-TmaYidC8HIS
(kDa)
83
62
47.5
(kDa)
80
60
50
NEB Express is superior to MC1061
Anti-His tag immunoblot
New England Biolabs
NEB Express is a BL21 derivative
+5+1
-2 0
Mitochondrial Oxa1p
C
+15
131
148
217
201
247
263
291
276 310
293
N+1
247 43
YidC-Oxa1p chimera functions in E. coli van Bloois et al. J. Biol. Chem. 280 (2005)
E.coli YidC
New England Biolabs
Mitochondrial Oxa1p
8xHIS
131
148
217
201
247
263
291
276 310
293
CBD
p8
N 43
Expression of p8CBDek-Oxa8HIS
complements the loss of E.coli YidC
ek site
New England Biolabs
Enterokinase cleavage of p8CBDek-Oxa8HIS
Full-length fusion (monomer)
anti-p8immunoblot
tetramer
p8CBD
6050
200140100 80
40
30
20
Enterokinase:
New England Biolabs
OXA1 8HISpVIII CBD
EK
Ptac FLAGp8CBDek-OXA8HISTM2
EK siteoptional
rbsH
OXA1 8HISpVIII CBDPtac
p8CBD-OXA8HISTM2rbsO
Acc65I – EcoRI – BamHI – SalI – HindIII – BsiWIpolylinker sites
Improved rbs sequence
New England Biolabs
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
L 1 2 3 4 5 6 7 8 9 10 11 12
rbs O rbs H rbs O rbs H
3 hr expression 20 hr expression
Oxa1pfusion
60
50
40
weaker rbsH results in higher protein expression at 20 hrs
Each series = 0, 40 or 400uM IPTG
New England Biolabs
NEB Expressproducing p8CBD-Oxa1p
0
0.5
1
1.5
2
0 5 10 15 20 25
rbsOrbsH
OD600
time (hrs)
Cells induced to express protein from rbsH continue to grow
NEB Expressproducing p8CBD-Oxa1p
IPTG = 400uM
New England Biolabs
L 1 2 3 4 5 6 7 8 9 10 11 12
rbs O rbs H rbs O rbs H
3 hr expression 20 hr expression
Oxa1pfusion
60
50
40
weaker rbsH results in higher protein expression at 20 hrs
Each series = 0, 40 or 400uM IPTG
New England Biolabs
NEB Expressproducing p8CBD-Oxa1p
++
++
CBD
fusion junction
cytoplasm
periplasm+----
N
+ +++- -
-
inner membrane
p8 TM2
FLAG/ek site
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
New England Biolabs
MP of interestpVIII-CBD
Enterokinase site
pT7 or Ptac
p8CBDek
E. coli expression vector p8CBDek
lacIqAmpR
orKanR
New England Biolabs