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Better Enzymes for Biosensors

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. Biosensors Bioremediation Diagnostics.

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Better Enzymes for Biosensors

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  1. 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

  2. Biosensors Bioremediation Diagnostics Bioinformatics Biocatalysis Protein Engineering Recombinant Protein Expression Peroxidase Transgenics Therapeutics

  3. Signals from HRP/SBP Reactions • Electrochemical • Colorimetric • Fluorimetric • Luminescent

  4. Protein Stabilization Strategies

  5. 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

  6. 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

  7. pBR_I 4.4 Kb 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

  8. 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

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

  10. Double Lys Lys 232 Lys 241 Lys 174 WT N E A N E A F A F Q N K232F/K241N Compare Lysine Mutants’ t1/2 Values

  11. Consensus Mutants Thermal Properties k(min-1) 0.056 0.054 0.085 0.068 0.051 0.065 0.078 ?

  12. 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

  13. Consensus Mutant T110V - shows a 25x increase in H2O2 stability- Unexpected bonus T102A COMBO T110V Q107N I180F Q106R WT

  14. C50 (mM) Q/Q N/N F/N WT E A F N Q N N A A N E E F E238 E239 K174 K232/241 K232 K241 ResultsH2O2 Stability, Rational Approach.

  15. 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 Lys: Conservative sub’. • number modifiable Lys. rHRP Directional Immobilization:Mutant Selection. Method: Rational Approach. • 21 Arg Residues in wt HRP • Achieve directional • immobilization by judicious residue selection?

  16. 0 ⅓ 3 18 1 2 4 Wild Type 0 New Lys + Retain 232, 241 New Lys + Remove 232, 241 Directional rHRP Immobilization:Proof of Principle Spot immobilization onto polyethersulfone membrane 30 pM HRP immobilized, DAB stained.

  17. 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! HRP .v. SBP

  18. 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?

  19. 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 …

  20. Chemically Modify SBP Carbohydrates

  21. Ferrocenylation of SBP (n = 3)

  22. 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.

  23. 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.)

  24. 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

  25. 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)

  26. Detailed SBP Conclusions • SBP deposited in microcavity etched at tip of a Pt microelectrode canperform 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

  27. 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. • 2007Bioconjugate Chem18:524-529. • 2006 Patent Application EP 06394027.4 • 2006 Trends Biotech 24: 355-363.

  28. 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

  29. 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)

  30. Acknowledgments • Emerging Technologies Conference Organizers • UMASS Lowell hosts • You, the Audience • Thank you for your attention

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