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Better Enzymes for Biosensors
Or, A Tale of Two Saucy
Little Peroxidases
Or, Improving Proteins With
New Tools & Old
Ciarán Ó’Fágáin
School of Biotechnology & National Centre for Sensor Research,
Dublin City University, Dublin 9, Ireland
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Biosensors
Bioremediation
Diagnostics
Bioinformatics
Peroxidase
Biocatalysis
Transgenics
Therapeutics
Protein
Engineering
Recombinant
Protein
Expression
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Signals from HRP/SBP Reactions
Fe 3+ HRP + H 2 O2
(resting)
k1
• Electrochemical
• Colorimetric
• Fluorimetric
• Luminescent
Compound I + H 2 O
(Fe 4+ =O
+porphyrin cation radical)
A.
AH
k3
k2
AH
A.
k4
Compound II
(Fe4 + = O)
H2 O2
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Compound III
(Fe 2+ .O 2 )
H2 O
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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”
PROTEIN
ENGINEERING
Reagents,
Many in vitro
Crosslinkers applications
Directly alters
protein
Cloned
gene, GM
expertise
Permanently
alters protein
“New Tools”
Applications
in vitro, in
vivo
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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
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Reaction of HRP Lysines with EGNHS
Crosslink
Lys174:
~20 % modified
Lys232:
{Lys241:
100 % modified
Lys65
No
Lys84
Biotech Bioeng 2001
76: 277-284
Lys149
80 % modified
}
significant
modification
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Our PelB-Wildtype rHRP-His6 Construct
• 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
pBR_I
4.4 Kb
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Mutations to Probe / Increase HRP Stability
• Rationally 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 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
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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
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Compare Lysine Mutants’ t1/2 Values
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Double Lys
Lys 232
t 1/2 at 50 oC (mins)
30
25
20
Lys 241
Lys 174
15
10
5
WT
N
E
A
N
E
A
F
A
F
Q
N
K232F/
K241N
0
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Consensus Mutants Thermal Properties
Mutant
t½ (min)
k (min-1)
Wildtype
12.4
0.056
T102A
12.9
0.054
Q106R
8.1
0.085
Q107D
10.3
0.068
T110V
13.7
0.051
I180F
Combined
10.7
8.8
0.065
?
0.078
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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
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Consensus Mutant T110V
- shows a 25x increase in H2O2 stability
- Unexpected bonus
430
420
C50 (mM)
410
400
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COMBO
I180F
T110V
Q107N
0
WT
5
Q106R
10
T102A
15
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Results
H2O2 Stability, Rational Approach.
300
275
250
225
175
70
60
50
40
30
20
0
WT
N
E
K174
A
N
E
A
K232
F
N
Q
E239
10
E238
C50
(%v/v)
C
50 (mM)
200
N
E
A
K241
F
Q/Q
N/N
F/N
K232/241
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rHRP Directional Immobilization:
Mutant Selection.
Method: Rational Approach.
• 21 Arg Residues in wt HRP
Lys: Conservative sub’.
•
number modifiable Lys.
• Achieve directional
immobilization by judicious
residue selection?
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 Helix
19, 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
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Directional rHRP Immobilization:
Proof of Principle
Spot immobilization onto polyethersulfone membrane
30 pM HRP immobilized, DAB stained.
⅓
0
Wild Type
1
2
3
4
18
0
New Lys +
Retain 232,
241
New Lys +
Remove 232,
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HRP .v. SBP
• HRP is moderately
heat stable
• SBP is notably heat stable,
moreso than HRP
• Chemical modification • Attempts to further
of HRP Lys increases
increase SBP heatstability
heat stability &
by chemically modifying
tolerance of solvent,
polypeptide yielded little
pH extremes
– SBP lacks exactly those Lys
that are targets in HRP!
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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
own ferrocene mediator?
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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 …
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Chemically Modify SBP Carbohydrates
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Ferrocenylation of SBP
Enzyme
Activity
(%)
Protein
(g/mL)
(n = 3)
Native SBP
100
439  34
Aminated SBP 128  14 234  24
FCA-SBP
67  14
238  38
No. free
–NH2
Iron
(ppb)
3  0.5
49  8
35  6
52  8
3  0.9
138  14
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CVs of bare electrode .v. both SBPs
•Innermost curve
(purple): No SBP in
electrode cavity
10.0
Current (nAmp)
.
7.5
5.0
2.5
•Middle curve
(black): native SBP
0.0
-2.5
-5.0
•Outermost curve
(red): FCA-SBP.
-7.5
-10.0
-12.5
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Potential (V), vs Ag/AgCl
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Native .v. Ferrocenylated SBP
Current response of
native (lower curve)
& ferrocenylated
SBP to successive
injections 2.5mol
H2O2. (Electrode
poised at -0.100 V.)
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Summary Conclusions – HRP & SBP
 Chemical modification can increase HRP thermal stability
 but not that of SBP
 Chem Mod (CM) CAN improve SBP’s biosensor properties,
however
 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
biosensor-friendly enzymes better for biosensors
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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)
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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
 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
Bioconjugate Chem (2007) 18: 524-529
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Recent HRP/ SBP publications
http://doras.dcu.ie/view/people/=D3=27F=E1g=E1in,_Ciar=E1n.html
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.
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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
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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)
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Acknowledgments
• Emerging Technologies Conference
Organizers
– UMASS Lowell hosts
• You, the Audience
– Thank you for your attention
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