The UK’s European university

The Tat pathway as a biotechnological tool for the expression and export of heterologous proteins in E. coliDr Kelly Walker & Professor Colin Robinson

k.l.walker@kent.ac.uk

Aims: to develop a new means of producing

recombinant proteins in E. coli

• global therapeutic protein market = ca.$100 billion p.a.

• over 1/3 of all approved proteins are made in E. coli

Currently there are 3 widely used expression methods:

• as soluble protein in cytoplasm

• refolding from inclusion bodies

• export to periplasm

An overview of protein targeting in bacteria

• Facilitates downstream

processing

• Enables disulphide bond

formation

Current model of Tat export in E. coli

2 3

1 binding to TatBC complex

recruitment of translocation

TatA complex of substrate

+ TatB TatC TatA

DpH

Advantages of the Tat pathway

• Can export difficult 'Sec-incompatible' proteins

e.g. GFP

• Not only capable of exporting folded proteins, Tat

preferentially exports correctly folded proteins i.e.

has an inbuilt quality control system

folded = export-competent

misfolded = 100% rejected

This ensures high product quality + minimal

heterogeneity

PreMat

Pre

Pre

Pre

30kDa

30kDa

30kDa

30kDa

46kDa

46kDa

46kDa

46kDa

WTΔtatABCDE

TCCMPTCCMP

YedY

YedY-Cys

YedY-Glu

YedY-Arg

Only WT YedY

is exported

Tat has a unique and exploitable proofreading

capacity

Proofreading works with proteins that don’t

actually exist (in nature)

Maquette (Les Dutton & Neil Hunter)

• Synthetic 4 α-helical protein

• Designed for electron transfer

• Interest because various residues or

cofactors can be ‘slotted’ in

BT6

• BT6 binds a heme.

• Heme binding stabilise structure by

up to 50%.

The Heme binding mutant is not exported

*

C M P C M P C M P

-------------- ----------------- ----------------

TorA-BT6 KK mutant Heme binding

mutant

Heme stabilised Heme cannot bind

Alex Jones

Jsaone@hotmail.com

Exploitation for

biotherapeutic production

Proof of Concept: high-Level export of GFP in

fed-batch fermentation

(Matos et al., Biotech Bioeng 2012)

M = medium

P = periplasm

Sp = spheroplasts

0

400

800

1200

1600

0 7 19 25 31 44 48

Time post induction (hrs)

mg

/L G

FP

.

0

40

80

120

160

200

OD

600

Periplasm Cells OD600

Yield = 1.2 g/l

Export of disulphide-bonded proteins

• Many biotherapeutics have disulphide bonds

• Tat will not export some proteins because they do not

fold correctly in the reducing cytoplasm (incorrectly

folded protein = rejection)

• Started collaboration with Lloyd Ruddock (Oulu) to use

new strains that DO form disulphide bonds in cytoplasm

Export of disulphide-bonded proteins

‘CyDisCo’ strains express thiol oxidase Erv1p + PDI in the

E. coli cytoplasm i.e. enables efficient disulfide bond

formation in cytoplasm

Can we persuade Tat to export prefolded, disulphide

bond containing proteins by attaching a TorA signal

peptide?

2 disulphide bond containing proteins: PhoA

and AppA are exported in CyDisCo+ cells

Erv1/PDI: - + - + - + - + ---------------------------------------------- spAppA spPhoA spAppA spPhoA

Spheroplasts Periplasm

Pre- - Mat -

pre-scFv- - Mat -

RR KK RR KK RR KK

Tat: + ++ + ++ + ++ + ++ + ++ + ++

- 25

- 55

- 15

-------- -------- -------- ------- ------------ -----------

Spheroplasts Periplasm Periplasm

A

B

BlotsCoomassie

Matos et al, 2014, Biotech Prog 30, 281-290

AppA = 4dsb, PhoA = 2dsb

Exported PhoA & AppA are active

Periplasmic activity

Matos et al, 2014, Biotech Prog 30, 281-290

Export of IFN α2β and hGH by Tat CyDisCo+

cells

- hGH

- IFN

30 - 23 -

17 -

CMPCMP ------------- ---------------

IFN hGH

CMPCMP ------------- ---------------

IFN hGH

+ CyDisCo Wild type cells

- hGH

- IFN

TorA-IFN

Alanen et al. (2015) Biochim Biophys Acta – Mol Cell Res

Wehaveexportedanumberofdifferentproteins..

Protein Size(kDa) NumberofDisulphideBonds

Exportefficiency(%)

scFv1 29 2 60

scFv2 27 2 75

hGH 24 2 90*

IFN 21 2 90*

VHdomain 9.5 0 90

hC-GSF 20 2 20

AppA 53 4 40

PhoA 51 2 40

LDH(dimer) 72 0 30

..andarenowmovingontomorecomplexmolecules

Tat-CyDisCo+ strains grow well in fed-batch

fementation

Export of hGH & IFN during fed-batch

fermentation

30 - 23 -

17 -

30 -

23 -

17 -

Time (h): 0 12 15 18 21 24 36

- hGH

- IFN

Yields of periplasmic hGH, IFN = 0.5 – 1 g/L

hGH = most abundant protein in periplasm after 4 h induction

MkCMPCMPCMP------------------------------------Un4h5h

-hGH

-hGH

>

> >

WT cells

TatExpress cells

New Technology: ‘Tat Express’ strains overexpress

Tat from the chromosome

Summary

1. The Tat pathway has a unique proofreading ability

(only correctly folded proteins are exported)

2. It can be used to export disulphide bonded proteins

(including pharmaceuticals)

3. The activities of the Tat translocon remain consistent

throughout fermentation (>60hours)

Future work:

1. To finish testing the capabilities of ‘Tat Express’ strains

(including with new signal peptides)

Acknowledgments

And the people who do the work:

THE UK’S EUROPEAN UNIVERSITY

www.kent.ac.uk

Small-scale bioreactor experiments at UCL

Fermenter operating conditions:

• Feed: 80% glycerol, 1.6 mL h-1

• pH: 6.95 ± 0.05

• Temperature: 30 ± 1 °C → 25 °C

• Gas flow rate: 100 vvm (air with

O2 when required)

• Impeller speed: 500 - 1100 rpm

• Working volume: 900 mL