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

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