Better Enzymes for Biosensors

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Better Enzymes for Biosensors
Or, A Tale of Two Saucy Little Peroxidases Or,
Improving Proteins With New Tools Old

• Ciarán ÓFágáin
• School of Biotechnology National Centre for
  Sensor Research,
• Dublin City University, Dublin 9, Ireland

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Biosensors Bioremediation Diagnostics
Protein Engineering Recombinant Protein
Expression
Bioinformatics Biocatalysis
Peroxidase
Transgenics Therapeutics
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Signals from HRP/SBP Reactions

• Electrochemical
• Colorimetric
• Fluorimetric
• Luminescent

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Protein Stabilization Strategies
TECHNIQUE NEEDS APPLICATIONS MERITS
IMMOBILIZN 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
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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
Lys174 20 modified Lys232 100
modified Lys241 80 modified Lys65
No Lys84 significant Lys149
modification
Crosslink

Biotech Bioeng 2001 76 277-284
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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

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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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Consensus Mutants Thermal Properties
k (min-1) 0.056 0.054 0.085 0.068 0.051 0.065 0.07
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Mutant t½ (min)
Wildtype 12.4
T102A 12.9
Q106R 8.1
Q107D 10.3
T110V 13.7
I180F Combined 10.7 8.8
?
 
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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
T102A
COMBO
Q107N
T110V
I180F
Q106R
WT
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ResultsH2O2 Stability, Rational Approach.
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rHRP Directional ImmobilizationMutant Selection.
Method Rational Approach.

• 21 Arg Residues in wt HRP

• Achieve directional
• immobilization by judicious residue selection?

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Directional rHRP ImmobilizationProof of
Principle
Spot immobilization onto polyethersulfone
membrane
30 pM HRP immobilized, DAB stained.
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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!

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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) 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
 (n 3)
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CVs of bare electrode .v. both SBPs

• Innermost curve (purple) No SBP in electrode
  cavity
• Middle curve (black) native SBP
• Outermost curve (red) FCA-SBP.

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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 SBPs 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 gt10X
• FCA-SBP is 3.5X more sensitive than native SBP.
  
• Linear current response to injected H2O2 betw.
  2.5 lt H2O2 lt 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/D327FE1gE1in,_CiarE1n.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 OBrien 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