School of Biotechnology & National Centre for Sensor Research 1 Better Enzymes for Biosensors...

1School of Biotechnology & National Centre for Sensor Research

Better Enzymes for Better Enzymes for BiosensorsBiosensors

Ciarán Ó’FágáinSchool of Biotechnology & National Centre for Sensor Research,

Dublin City University, Dublin 9, Ireland

Or, A Tale of Two Saucy Or, A Tale of Two Saucy Little PeroxidasesLittle Peroxidases

Or, Improving Proteins With New Tools & Old


Peroxidase

BiosensorsBiosensors

Bioremediation Diagnostics

Protein Engineering

Recombinant Protein Expression

Bioinformatics

Biocatalysis

Transgenics Therapeutics


Signals from HRP/SBP Reactions

• Electrochemical

• Colorimetric

• Fluorimetric

• Luminescent

Fe 3+ HRP + H 2 O 2 Compound I + H 2 O

(resting) k 1 (Fe 4+ =O

+porphyrin cation radical)

A. AH

k 3 k 2 AH A.

k 4 Compound II Compound III

(Fe 4 + = O) (Fe 2+ .O 2 )

H 2 O 2 H 2 O


Protein Stabilization Strategies

TECHNIQUE NEEDS APPLICATIONS

MERITS

IMMOBILIZ’N Solid phase, Many links

Bioreactors,biosensors,diagnostics

Widely used,Many types of support

ADDITIVES Osmolytes, Excipients

Long-term storage

Effective, protein itself is unaltered

CHEMICAL MODIFICATION

“Old Tools”

Reagents, Crosslinkers

Many in vitro applications

Directly alters protein

PROTEIN ENGINEERING

“New Tools”

Cloned gene, GM expertise

Applications in vitro, in vivo

Permanently alters protein


Chemical Modification of HRP Lys

• EGNHS [Ethylene glycol bis-(succinimidyl succinate)]– Homobifunctional crosslinker– Spans up to 16 Å– Neutralizes +ve charge of Lys

• Acetic acid N-hydroxy succinimide ester– Non-crosslinking monofunctional– Acts like EGNHS

• Phthalic anhydride– Introduces bulky aromatic

group– Reverses +ve charge of Lys


Reaction of HRP Lysines with EGNHS

Lys174: ~20 % modified

Lys232: 100 % modified

Lys241: 80 % modified

Lys65 No

Lys84 significant

Lys149 modification

}Biotech Bioeng 2001

76: 277-284

Crosslink {


Our PelB-Wildtype rHRP-His6 Construct

pBR_I

4.4 Kb

• Recombinant HRP: Problems• Inclusion bodies

• Tricky to refold• Hyperglycosylation in yeast• Low yields from E. coli• 1999: Arnold describes soluble HRP recombinant• 2002: donation to DCU


Mutations to Probe / Increase HRP Stability

• Rationally designed mutationsRationally designed mutations based on our “prior art”:mutate Lys 174, 232 & 241, observe effects on stability.• (directed evolution study published but no previous SDM dealing with HRP stability)

• Semi-rational DesignSemi-rational Design: “Consensus Approach” to identify potential mutations.

• Compare amino acid sequences of related proteins to identify the ‘consensus’ amino acid at any position

• Postulate that the ‘consensus’ amino acid contributes more to stability than rarer ones.

• downloaded & aligned 100 plant peroxidase sequences


Rational HRP Mutant Selection

• Rational approach to mutation of key (+vely charged) Lys residues 174, 232, 241.

Ala (A) Small, non-polar

Asn, Gln (N, Q) Polar, uncharged

Glu (E) Charge reversal

Phe (F) Bulky, hydrophobic


0

5

10

15

20

25

30

35

t 1

/2 a

t 50

oC

(m

ins)

WT E N E A N NF A F Q K232F/K241NA

Lys 174

Lys 232

Lys 241

Double Lys

Compare Lysine Mutants’ t1/2 Values


Consensus Mutants Thermal Properties

MutantMutant tt½½ (min)

Wildtype 12.4

T102A 12.9

Q106R 8.1

Q107D 10.3

T110V 13.7

I180FCombined

10.78.8

kk (min-1)

0.056

0.054

0.085

0.068

0.051

0.065

0.078

?


Scarcely any differences!

• Very disappointing outcome– No improvements in thermal stability– No enhanced solvent tolerances– No catalytic differences

• At least, with ABTS substrate

– Why such poor results?• Literature shows that ‘consensus’ works for other enzymes• Alpha-helix scaffold seems conserved in plant peroxidases

•Try something else: oxidative stability–Excess H2O2 substrate (oxidant) can inactivate HRP


Consensus Mutant T110V - shows a 25x increase in H2O2 stability

- Unexpected bonus

C5

0 (

mM

)

0

5

10

15

20

400

410

420

430

T102A

Q107N

T110V

I180F

COMBO

WT

Q106R


C5

0 (%

v/v

)

0

10

20

30

40

50

60

70

175

200

225

250

275

300

K174 K232 K241 K232/241E23

8

E23

9

N E A N E A F N E A F Q/Q N/N F/NQNWT

C50

(m

M)

ResultsH2O2 Stability, Rational Approach.


rHRP Directional Immobilization:Mutant Selection.

Method: Rational Approach.

• 21 Arg Residues in wt HRP

• Achieve directional immobilization by judicious residue selection?

Lys: Conservative sub’.

• number modifiable Lys.

Possible Arginine Residues.19, 27, 31, 38, 62, 75, 82, 93, 118, 123, 124, 153, 159, 178,

183, 206, 224, 264, 283, 298, 302.

Located in Helix19, 27, 38, 75, 82, 93, 123, 124, 153, 206, 264.

Located in Protein Core.31, 183, 298.

Similar Plane as Active Site Entrance.62, 178, 224, 302

Arginine Residues Selected for Mutation.118, 159 and 283


Directional rHRP Immobilization:Proof of Principle

Spot immobilization onto polyethersulfone membrane

30 pM HRP immobilized, DAB stained.

Wild Type

New Lys + Retain 232, 241

New Lys + Remove 232, 241

0 ⅓ 1

0

2 3 4 18


HRP .v. SBP

• HRP is moderately heat stable

• Chemical modification of HRP Lys increases heat stability & tolerance of solvent, pH extremes

• SBP is notably heat stable, moreso than HRP

• Attempts to further increase SBP heatstability by chemically modifying polypeptide yielded little– SBP lacks exactly those Lys

that are targets in HRP!


A Recurring Issue in Biosensors

• Electron transfer from enzyme active site to electrode can be inefficient & rate-limiting: may need to add external mediator (such as ferrocene) to bridge the distance.

• Sugars of glycoproteins can increase the enzyme-electrode distance: undesirable.– Use sugar-free recombinant proteins ex E. coli ?

• Why not alter protein so that it carries its Why not alter protein so that it carries its own ferrocene mediator?own ferrocene mediator?


Ferrocene carboxylic acid

• … is available & can be coupled to free –NH2 via carbodiimide BUT …

• SBP is poor in reactive –NH2, so need to add on extra –NH2 groups to enable attachment of ferrocene carboxylic acid (FCA)

• One possible way of doing this is to …


Chemically Modify SBP Carbohydrates


Ferrocenylation of SBP

(n = 3)

Enzyme Activity (%)

Protein (g/mL)

No. free –NH2

Iron (ppb)

Native SBP 100 439 34 3 0.5 49 8

Aminated SBP

128 14 234 24 35 6 52 8

FCA-SBP 67 14 238 38 3 0.9 138 14


CVs of bare electrode .v. both SBPs

-12.5

-10.0

-7.5

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

0.10.20.30.40.50.60.7

Potential (V), vs Ag/AgCl

Cur

rent

(nA

mp)

.

•Innermost curve (purple): No SBP in electrode cavity

•Middle curve (black): native SBP

•Outermost curve (red): FCA-SBP.


Native .v. Ferrocenylated SBP

Current response of native (lower curve) & ferrocenylated SBP to successive injections 2.5mol H2O2. (Electrode poised at -0.100 V.)


Summary Conclusions – HRP & SBP Chemical modification can increase HRP thermal stabilityChemical modification can increase HRP thermal stability

but not that of SBP

Chem Mod (CM) CANCAN improve SBP’s biosensor properties, however

Genetic manipulation (GM) is also powerful …Genetic manipulation (GM) is also powerful …

Single substitutions increase HRP resistance to excess H2O2 …

… while other mutations permit its orientated immobilization.

So, both old (CM) & new (GM) tools can make these So, both old (CM) & new (GM) tools can make these biosensor-friendly enzymes better for biosensorsbiosensor-friendly enzymes better for biosensors


Detailed Conclusions - rHRP

• HRP mutants K232F, K232N, K232F/K241N show modest increase in stability to heat (at 50oC)

• Consensus mutant T110V & Lys mutants K232N, K241F, K232N/K241F, K232N/K241N are notably more tolerant of H2O2 than wild type– Increased oxidative/ chemical stability

• Can achieve orientated/ directional immobilization of HRP by mutations R118K/R153K/R283K – (plus K232N/K241F)


Detailed SBP Conclusions• SBP deposited in microcavity etched at tip of a Pt micro electrode can perform direct, mediator-free electron transfer

• Can covalently bind ferrocene (FCA) mediator to SBP glycans

• ~1.5 ferrocenes/SBP molecule, effective even with crude SBP

Bioconjugate Chem (2007) 18: 524-529

FCA-SBP outperforms native SBP in etched Pt electrode

Enzyme-electrode electron transfer rate increases >10X

FCA-SBP is ~3.5X more sensitive than native SBP.

Linear current response to injected [H2O2] betw. 2.5 < [H2O2] < 42.5 M.

These microcavity sensors have potential as reagentless electrodes to measure H2O2 & other analytes that act as electron donors for peroxidases


2008 Biochimie 90: 1414-1421. 2008 Biochimie 90: 1389-1396. 2007 BMC Biotechnology 7: 86.2007 Biochimie 89: 1029-1032. 2007 Bioconjugate Chem 18: 524-529.2006 Patent Application EP 06394027.4 2006 Trends Biotech 24: 355-363.

Recent HRP/ SBP publicationsRecent HRP/ SBP publications

http://doras.dcu.ie/view/people/=D3=27F=E1g=E1in,_Ciar=E1n.htmlhttp://doras.dcu.ie/view/people/=D3=27F=E1g=E1in,_Ciar=E1n.html


Acknowledgments – GM work• Materials

– FH Arnold, Caltech, USA (HRP gene)

• Finance & Personnel– IRCSET* & DCU RAP Pgrad Award (Barry

Ryan) – DCU RAP Albert College Award (CÓF)

• Advice & Expertise– Drs P Clarke, P Leonard, P Ó Cuív, P-R Vaas,

C Viguier, J Finlay, S Hearty*Irish Research Council for Science, Engineering & Technology


Acknowledgments – CM work• Personnel

– Orlaith Ryan Enzyme Microb Tech (1994) 16: 501-505

– Enda Miland Enzyme Microb Tech (1996) 19: 63-67

– Anne-Marie O’Brien Biotech Bioeng (2003) 81: 233-240

– Neil Carolan Bioconjugate Chem (2007) 18: 524-529

• Finance– Amersham Intl, British Council, Dublin City Univ, EC

Framework 4 (BIO-CT97-2031), Eolas, Fingal County Council.

• Advice & Expertise– AT Smith, KG Welinder, PF Nielsen, MR Smyth (HRP) – RJ Forster (SBP electrochemistry)


Acknowledgments

• Emerging Technologies Conference Organizers– UMASS Lowell hosts

• You, the Audience– Thank you for your attention

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