Smart Materials: Development of Protein Recognition Polymers as Novel Surfaces for Protein Crystallisation.
Lead Research Organisation:
University of Surrey
Department Name: Health and Medical Sciences
Abstract
Our proposed research investigates a new approach to the use of a novel class of protein recognition polymers (PRPs) as surfaces for inducing protein crystallisation.Protein crystallisation is vital to the Success of the Human Genome project which has opened up exciting opportunities for the treatment of disease. However, it is often not the genes themselves that are the targets of potential drugs, but the thousands of proteins encoded by these genes. In order to understand how proteins perform their various tasks, we need to know the structure of these proteins. The most effective technique for determining protein structure is X-ray crystallography which requires high quality crystals of the proteins. The current structural genomics/proteomics projects worldwide have set out to determine the structures of 100,000 proteins but in spite of investing considerable funds and effort, they have had limited success because obtaining crystals is a major bottleneck to progress. There is an urgent need for new and improved ways to enable the growth of crystals.The problem lies in either getting no crystals at all, or even more frustrating - getting crystals that are not of a high enough quality to allow structure determination. The ultimate means to obtain good crystals is to control their conception stage, i.e. the nucleation stage which is the first step that determines the entire crystallization process. This is not an easy task hence the Holy grail is to find a 'universal nucleant' - a substrate that would induce crystallization of any protein. Several substances such as minerals, human hair, silicas have been used as nucleants, but with limited success. PRPs can selectively recognize particular proteins and therefore are very likely to serve as the ideal nucleants because rather than use random materials as nucleants, it will be possible to prepare PRPs for each protein while it is in solution and then get it to crystallise using that PRP. The results of the protein crystallisation studies will enable optimization of the PRPs for general protein capture applications (e.g. protein extraction and protein biosensing). The PRP technology is still in its infancy and proper characterisation methods do not currently exist. The nucleation studies will allow an interesting route to the feedback of essential information regarding whether parameters such as crosslinking density and choice of monomers can lead to better PRP formation. Biosensors for proteins are currently expensive to develop because they require the use of expensive antibodies. Because of the biomimicry capabilities (and their potential to act as synthetic antibodies), PRPs potentially offer a route to the development of new low-cost biosensors for cancer markers and bioterrorism markers.
People |
ORCID iD |
| Subrayal Reddy (Principal Investigator) |
Publications
EL-Sharif H
(2015)
Spectroscopic and quartz crystal microbalance (QCM) characterisation of protein-based MIPs
in Sensors and Actuators B: Chemical
EL-Sharif H
(2014)
Advanced Synthetic Materials in Detection Science
El-Sharif HF
(2014)
Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning.
in Analytica chimica acta
EL-Sharif HF
(2014)
Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).
in Physical chemistry chemical physics : PCCP
Khurshid S
(2015)
Automating the application of smart materials for protein crystallization.
in Acta crystallographica. Section D, Biological crystallography
Reddy S
(2013)
Protein detection using hydrogel-based molecularly imprinted polymers integrated with dual polarisation interferometry
in Sensors and Actuators B: Chemical
Reddy S
(2011)
Electrochemical probing of selective haemoglobin binding in hydrogel-based molecularly imprinted polymers
in Electrochimica Acta
Reddy SM
(2012)
Protein crystallization and biosensor applications of hydrogel-based molecularly imprinted polymers.
in Biomacromolecules
Saridakis E
(2011)
Protein crystallization facilitated by molecularly imprinted polymers.
in Proceedings of the National Academy of Sciences of the United States of America
| Description | We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as "smart materials," are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8-10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates. We have characterized the imprinting capability of a family of acrylamide polymer-based molecularly imprinted polymers (MIPs) for bovine hemoglobin (BHb) and trypsin (Tryp) using spectrophotometric and quartz crystal microbalance (QCM) sensor techniques. Bulk gel characterization on acrylamide (AA), Nhydroxymethylacrylamide (NHMA), and N-isopropylacrylamide (NiPAM) gave varied selectivities when compared with control polymers. We have also harnessed the ability of the MIPs to facilitate protein crystallization as a means of evaluating their selectivity for cognate and noncognate proteins. Crystallization trials indicated improved crystal formation in the order NiPAM < AA < NHMA. QCM studies of thin film MIPs confirm this trend with Nhydroxymethyl acrylamide MIPs exhibiting best discrimination between MIP and NIP and also cognate/noncognate protein loading. Equivalent results for acrylamide MIPs suggested that the cavities were equally selective for both proteins, while N-isopropylacrylamide MIPs were not selective for either cognate BHb or noncognate BSA. All BHb MIP-QCM sensors based on AA, NHMA, or NiPAM were essentially nonresponsive to smaller, noncognate proteins. Protein crystallization studies validated the hydrophilic efficacy of MIPS indicated in the QCM studies. |
| Exploitation Route | Through Imperial Innovations, we are marketing MIP crystallization kits (a joint venture between University of Surrey and Imperial College, London). |
| Sectors | Agriculture Food and Drink Chemicals Education Environment Healthcare |
| URL | http://www.imperialinnovations.co.uk/license/available-technologies/chayen-reddy-mip/ |
| Description | 1. Negotiations with companies to develop smart materials into a crystallization kit ongoing 2. Publication and new collaborations 3. Public communication to lay audiences (through stand-up comedy) to improve understanding and stimulate thought. |
| First Year Of Impact | 2011 |
| Sector | Chemicals,Education,Other |
| Description | NERC and RSC (ACTF) Analytical Studentship |
| Amount | £72,000 (GBP) |
| Funding ID | NE/J01/7671 |
| Organisation | Natural Environment Research Council |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2012 |
| End | 03/2016 |
| Description | Smart Materials: Hydrogel-based Molecularly Imprinted Polymers (HydroMIPs) to Facilitate Protein Crystallisation KTA APPLICATION |
| Amount | £22,572 (GBP) |
| Funding ID | EP/H00189/1 |
| Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 09/2011 |
| End | 03/2012 |
| Description | The Leverhulme Trust |
| Amount | £163,720 (GBP) |
| Organisation | The Leverhulme Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 07/2014 |
| End | 07/2017 |
| Description | The Royal Society_International Exchanges |
| Amount | £12,000 (GBP) |
| Funding ID | IE130745 |
| Organisation | The Royal Society |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 11/2013 |
| End | 12/2015 |
| Description | Wellcome Trust Seed Fund |
| Amount | £100,000 (GBP) |
| Funding ID | 108003/Z/15/Z |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 06/2016 |
| End | 12/2017 |
| Description | Integration of smart materials with pattern recognition and nanofabrication techniques for the development of novel electrochemical disease diagnostics. |
| Organisation | North Carolina State University |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | 1. Fabricate nanostructured electrodes that can be tethered to the monomers used in the production of the HydroMIPs. (NCSU) 2. Interrogate MIPs electrochemically in different protein loading states and demonstrate that pattern recognition techniques can discriminate between target and non-target proteins. (UK and USP) 3. Combine nanoelectrode-integrated MIPs with pattern recognition software to optimise and characterise HydroMIPs for the rational design of novel electrochemical HydroMIP-based disease diagnostics. (Surrey/NCSU/USP) Dr Reddy's Group and Dr Paixão's Group were heavily involved in characterising suitable MIPs and thereby demonstrating proof of concept that Dr Paixão's pattern recognition and principal component analysis techniques can be applied to demonstrate that MIPs can be selective for protein markers. MIPs were transferred to Prof Narayan's Lab (NCSU) for integration to conductive microneedle structures in the preparation and demonstration of novel nanosensors for disease markers. However, this aspect of the project did not yield any useful data in the timescale of the study. Issues surrounding the facile integration of MIPs to the microneedle structures. Results were also fed-back to the the partner Groups at fortnightly intervals (via Skype). The collaborative group prepared papers for conference presentation and publication as detailed in the next section. |
| Collaborator Contribution | The collaboration was initiated with Dr Paixão (5 day visit) and his post-doc and postgraduate student (21 days) visiting Dr Reddy's Lab in the UK. Dr Reddy's group showed how MIPs are produced and how they can be attached to electrochemical electrodes. Dr Paixão's students demonstrated their software on use of chemometrics and pattern recognition to produce data sets from raw cyclic voltammogram data. Dr Paixão's student and post-doc worked alongside Dr Reddy's student on applying the pattern recognition software to cyclic voltammogram data produced for MIPs in different loading states. Prof Narayan and his student will microneedle structures metallised with copper and gold for initial electrode testing. Dr Paixão took the opportunity in their short visit to give a seminar to academics and students at University of Surrey to expand potential future collaborations. |
| Impact | Papers: 1. L. Bueno, H. F. El-Sharif, M. O. Salles, R. D. Boehm, R. J. Narayan, T. R. L. C. Paixão and S. M. Reddy (2014) MIP-based electrochemical protein profiling. Sensors and Actuators B: Chemical, 204, pp. 88-95. 2. L Bueno, A Cottell, SM Reddy, TRLC Paixão (2015) Coupling dye-integrated polymeric membranes with smartphone detection to classify bacteria, RSC Advances, 5(119), pp. 97962-97965. 3. L. Bueno, G. Meloni. S.M. Reddy and T.R.L.C Paixao (2015) Use of plastic-based analytical device, smartphone and chemometric tools to discriminate amines, RSC Advances, 5, pp. 20148 - 20154. Books/Book Chapters 1) 'Advanced Synthetic Materials in Detection Science' S.M. Reddy (Ed.), RSC Publishing; ISBN: 9781849735933; Published Sept 2014. Conferences: 1) S.M.Reddy, Advances in Protein-based Molecularly Imprinted Polymers, Symposium on Materials for Biosensors - IMRCXXIII Congress, 2014, Cancun, Mexico, Aug 2014. (O; Invited) 2) S.M. Reddy, D. Stevenson, H. F. El-Sharif, Daniel M. Hawkins, Q. T. Phan. Smart materials: development of antibody-mimics based on molecularly imprinted polymers and their use in biosensors. International THERMEC' 2013 Conference, Las Vegas, USA, Dec 2013. (O; Invited) 3) Thiago Regis Longo Cesar Paixao, Maiara Oliveiera Salles, Ligia Bueno, William Reis de Arauji, Subrayal M. Reddy, Hazim F. EL-Sharif, Roger J. Narayan. Development of electronic tongues based on low cost fabrication process and modified electrodes aiming to on-site discrimination of forensic, clinical and food samples. International THERMEC' 2013 Conference, Las Vegas, USA, Dec 2013. (O; Invited) 4) Pittcon 2015: Cellulose Acetate Membrane-based Colorimetric Device to Discriminate Amines, Lígia Bueno, Subrayal M. Reddy, Thiago R.L.C. Paixão |
| Start Year | 2012 |
| Description | Integration of smart materials with pattern recognition and nanofabrication techniques for the development of novel electrochemical disease diagnostics. |
| Organisation | Universidade de São Paulo |
| Country | Brazil |
| Sector | Academic/University |
| PI Contribution | 1. Fabricate nanostructured electrodes that can be tethered to the monomers used in the production of the HydroMIPs. (NCSU) 2. Interrogate MIPs electrochemically in different protein loading states and demonstrate that pattern recognition techniques can discriminate between target and non-target proteins. (UK and USP) 3. Combine nanoelectrode-integrated MIPs with pattern recognition software to optimise and characterise HydroMIPs for the rational design of novel electrochemical HydroMIP-based disease diagnostics. (Surrey/NCSU/USP) Dr Reddy's Group and Dr Paixão's Group were heavily involved in characterising suitable MIPs and thereby demonstrating proof of concept that Dr Paixão's pattern recognition and principal component analysis techniques can be applied to demonstrate that MIPs can be selective for protein markers. MIPs were transferred to Prof Narayan's Lab (NCSU) for integration to conductive microneedle structures in the preparation and demonstration of novel nanosensors for disease markers. However, this aspect of the project did not yield any useful data in the timescale of the study. Issues surrounding the facile integration of MIPs to the microneedle structures. Results were also fed-back to the the partner Groups at fortnightly intervals (via Skype). The collaborative group prepared papers for conference presentation and publication as detailed in the next section. |
| Collaborator Contribution | The collaboration was initiated with Dr Paixão (5 day visit) and his post-doc and postgraduate student (21 days) visiting Dr Reddy's Lab in the UK. Dr Reddy's group showed how MIPs are produced and how they can be attached to electrochemical electrodes. Dr Paixão's students demonstrated their software on use of chemometrics and pattern recognition to produce data sets from raw cyclic voltammogram data. Dr Paixão's student and post-doc worked alongside Dr Reddy's student on applying the pattern recognition software to cyclic voltammogram data produced for MIPs in different loading states. Prof Narayan and his student will microneedle structures metallised with copper and gold for initial electrode testing. Dr Paixão took the opportunity in their short visit to give a seminar to academics and students at University of Surrey to expand potential future collaborations. |
| Impact | Papers: 1. L. Bueno, H. F. El-Sharif, M. O. Salles, R. D. Boehm, R. J. Narayan, T. R. L. C. Paixão and S. M. Reddy (2014) MIP-based electrochemical protein profiling. Sensors and Actuators B: Chemical, 204, pp. 88-95. 2. L Bueno, A Cottell, SM Reddy, TRLC Paixão (2015) Coupling dye-integrated polymeric membranes with smartphone detection to classify bacteria, RSC Advances, 5(119), pp. 97962-97965. 3. L. Bueno, G. Meloni. S.M. Reddy and T.R.L.C Paixao (2015) Use of plastic-based analytical device, smartphone and chemometric tools to discriminate amines, RSC Advances, 5, pp. 20148 - 20154. Books/Book Chapters 1) 'Advanced Synthetic Materials in Detection Science' S.M. Reddy (Ed.), RSC Publishing; ISBN: 9781849735933; Published Sept 2014. Conferences: 1) S.M.Reddy, Advances in Protein-based Molecularly Imprinted Polymers, Symposium on Materials for Biosensors - IMRCXXIII Congress, 2014, Cancun, Mexico, Aug 2014. (O; Invited) 2) S.M. Reddy, D. Stevenson, H. F. El-Sharif, Daniel M. Hawkins, Q. T. Phan. Smart materials: development of antibody-mimics based on molecularly imprinted polymers and their use in biosensors. International THERMEC' 2013 Conference, Las Vegas, USA, Dec 2013. (O; Invited) 3) Thiago Regis Longo Cesar Paixao, Maiara Oliveiera Salles, Ligia Bueno, William Reis de Arauji, Subrayal M. Reddy, Hazim F. EL-Sharif, Roger J. Narayan. Development of electronic tongues based on low cost fabrication process and modified electrodes aiming to on-site discrimination of forensic, clinical and food samples. International THERMEC' 2013 Conference, Las Vegas, USA, Dec 2013. (O; Invited) 4) Pittcon 2015: Cellulose Acetate Membrane-based Colorimetric Device to Discriminate Amines, Lígia Bueno, Subrayal M. Reddy, Thiago R.L.C. Paixão |
| Start Year | 2012 |
| Description | Smart Materials: Development of Metal-coded protein biosensors based on molecularly imprinted polymers |
| Organisation | Heriot-Watt University |
| Department | Chemistry |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | UK Benefits and Impact: Knowledge and materials transfer of key compound for integration into MIPs. This has opened up possibilities for improve MIP selectivity for non-metallo-proteins. The UK institute will benefit from the opening of new channels of research which could potentially lead to patentable technologies and SME interest in further developing the MIP technologies for food, environmental and biomedical applications. The collaboration will open up opportunities to apply for further international collaborative funding (e.g. from the Newton Fund initiatives). |
| Collaborator Contribution | India Benefits and Impact: Hitherto, The Indian group have knowledge on the synthesis of metal complexes; the UKIERI collaborative project has provided opportunity to gain knowledge in studying the bio-chemical applications of metal complexes by integrating the compounds into MIPS for selective characterization of metal-proteins based on variable oxidation state of metal in the complex. Collaboration provided opportunities to the faculty members of SV University for applying further international collaborative projects to RAEA and Newton Fund. |
| Impact | Papers: HF El-Sharif, H Yapati, S Kalluru, SM Reddy (2015) Highly selective BSA imprinted polyacrylamide hydrogels facilitated by a metal-coding MIP approach, Acta Biomaterialia, 28, pp. 121-127. Conferences: 1. Functional Nanomaterials in Industrial Applications: Academic-Industry Meet, University of Central Lancashire, Preston, UK (29th-31st March 2016); Invited talk. 2. Biomatsens 2016 - International Congress on Biomaterials and Biosensors, Istanbul, Turkey (1st-3rd June 2016); Invited talk. 3. Euro Biosensors and Bioelectronics Conference, Valencia, Spain (30th June-2nd July 2016) |
| Start Year | 2013 |
| Title | PROTEIN CRYSTALLIZATION USING MOLECULARLY IMPRINTED POLYMERS |
| Description | The invention relates to a method comprising: providing a molecularly imprinted polymer imprinted with a first peptide or protein;exposing said molecularly imprinted polymer to a supersaturated solution of a second peptide or protein;and forming a nucleus of, and/or growing a crystal of, said second peptide or protein on said molecularly imprinted polymer. |
| IP Reference | WO2010139972 |
| Protection | Patent application published |
| Year Protection Granted | 2010 |
| Licensed | Commercial In Confidence |
| Impact | Collaboration with other groups who wish to crystallize 'hard to crystallize' proteins in Brazil. Commercial interest in crystallization kit, now being marketed through Imperial Innovations. Patent administered at University of Surrey. See http://www.imperialinnovations.co.uk/license/available-technologies/chayen-reddy-mip/ |
| Description | Bright Club Guildford 2012 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | 100 people attended my stand-up comedy based on my research. I was invited to be on TV, and appeared on BBC4's Some Boffins with Jokes, first aired Summer 2013 and re-run Sept 2014 on BBC2. I also performed my research-based stand-up comedy at the Edinburgh Fringe Festival 2013 and was invited to perform at the Winchester Science Festival 2014. |
| Year(s) Of Engagement Activity | 2012 |
| URL | https://www.youtube.com/watch?v=wKMAwosmYlQ |
| Description | Bright Club Guildford 2013 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | 100 people listened to my stand-up comedy about Chemistry and Materials Science Appearance and performance at Edinburgh Fringe 2013 |
| Year(s) Of Engagement Activity | 2013 |
| URL | https://www.youtube.com/watch?v=StuA_THBfn8 |
| Description | Edinburgh Fringe 2013 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | 60 people attended my performance on Chemistry and Smart Materials research Not known |
| Year(s) Of Engagement Activity | 2013 |
| URL | https://en-gb.facebook.com/universityofsurrey/posts/10151685240158241 |
| Description | Winchester Science Festival 2014 |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Public/other audiences |
| Results and Impact | Communication of science and research through the medium of stand-up comedy. |
| Year(s) Of Engagement Activity | 2014 |