Bioorthogonal site-selective protein immobilisation and labelling

Recently, we discovered that an enzyme (PPTase) can be used to attach proteins, which have been modified with a small tag, to a range of different materials that present the naturally produced molecule coenzyme A. This method is mild and efficient and enables the site-specific attachment of proteins in a uniform orientation on to a surface, thus maintaining high activity. Given that the short tag attached to the protein is unique in structure, it is possible to immobilise proteins directly from cell extracts without the need for time-consuming isolation of the protein of interest in pure form. However, the efficiency of this immobilisation from cellular extracts is reduced due to the presence of the cells' own coenzyme A, which can compete for attachment to the tagged protein. In this project we will engineer PPTases that are more efficient and no longer recognise the natural coenzyme A, but instead are specific for man-made analogues. We envisage that the new PPTase enzymes will be ideal for the rapid attachment of large numbers of tagged proteins on to chips (glass slides) directly from complex cellular mixtures. Using such protein chips (or arrays) it is possible to characterise large numbers of proteins simultaneously on a single chip, with minute amounts of material. Indeed protein chips can be used to screen for other biomolecules that bind to the immobilised proteins, which can help address fundamental biological questions relating to protein function. Immobilised proteins can also be used for drug screening, diagnostics and as a part of detection devices. To date, protein arrays have been limited to relatively modest numbers of proteins. However it is estimated that in humans alone, there could be hundreds of thousands of unique proteins. The generation of future protein arrays, which might accommodate larger numbers proteins, requires highly efficient protein immobilisation methodology and further miniaturisation of protein features on the surface of chips. In light of this we will combine and exploit our new protein immobilisation method with state-of-the-art nanofabrication methods which allow nanometre chemical 'spots' (less than a billionth of a meter in diameter) to be generated on glass slides, on to which we will attach proteins. We will then demonstrate the activity of the proteins attached to the nanoscale features, by studying their interactions with other proteins, DNA and drug molecules. In addition to protein immobilisation the new more efficient PPTase enzymes we develop can also be used in the site-specific labelling of proteins. The attachment of fluorescent labels, drugs, and other molecules to proteins is also important for studying protein function and in the development of protein based drugs (biopharmaceuticals).

Currently the fabrication of protein arrays is hampered by challenges associated with high-throughput expression and purification of the large numbers of proteins and the lack of effective and simple methodologies that can be used to immobilise these proteins whilst retaining their activity/functionality. Furthermore, the number of features that can be written, addressed and detected on the surface, using standard technology remains small relative to the size of a proteome. We propose to develop an highly efficient method for site-selective immobilisation of proteins on to surfaces using an engineered variant of a phosphopantetheinyl transferase enzyme (PPTase). Our approach will be entirely bioorthogonal, allowing immobilisation of functional proteins, possessing a small peptide (12mer) tag, directly from cell lysates on to surfaces functionalised with coenzyme A (CoA) analogues, circumventing the need for protein purification. We will then demonstrate the application of this method, together with the relevant surface chemistries, by producing nano-scale protein features on surfaces that have been patterned using scanning near-field photolithography and dip-pen nanolithography. Model proteins of interest will be immobilised on to the nanopatterned surfaces and subjected to a range of assays to demonstrate the binding of protein partners, specific DNA sequences and small drug-like molecules. By combining site-selective protein immobilisation and nanolithography, we aim to develop new techniques that will underpin the future development of functional high-density protein arrays, which would be extremely valuable for proteomics research, as well as drug screening and diagnostic applications. Finally, to further underline the general utility of our methodology, we will also demonstrate how the bioorthogonal PPTase enzymes we have engineered can also be used for site-specific labelling of proteins.

WHO WILL BENEFIT: Many biotechnology companies produce and sell kits and other materials that can be used for protein immobilisation. Some of these companies [e.g. Invitrogen (ProtoArray), Promega (HaloLink Protein Array Systems), Sigma-Aldrich, Thermo-Scientific and ProtNeteomix] also use immobilisation technology to generate in-house protein arrays. In addition to academic labs, many pharmaceutical and biotechnology companies have begun to use immobilised proteins in drug screening.. Many hospitals, and other government funded medical labs could benefit from using immobilised proteins and antibodies as diagnostic tools, which are invaluable in fast detection of diseases using extremely small samples from patients (biopsy). There are also a number of companies that have commercialised related protein labelling technologies including: GeneCopoeia (AviTag), Innovabiosciences, AnaSpec (Eurogentec), New England Biolabs (SNAP- and CLIP-tags). HOW WILL THEY BENEFIT: We aim to develop new-engineered bioorthogonal enzymes which can be used for protein immobilisation and labelling. The approach that we will develop offers a combination of features that are not concurrently present in other methods (i.e. bioorthogonal, selective, covalent, non-toxic, employing a small genetically encodable tag that can be at either terminal or an exposed loop, requires no further post-expression derivatisation or purification) and would therefore be superior for applications requiring protein immobilisation such as biomolecular arrays or sensors. We will also develop compatible nanopatterning techniques and the associated surface as well as synthetic chemistry required for site-specific labelling. Any component or indeed the whole package of methodologies and materials we develop will be made available to industrial and other partners to further develop through appropriate collaborations and licensing agreements. For example companies that manufacture their own protein arrays may wish to utilise our immobilisation and surface chemistries within their own array platform, whilst others may choose to utilise our bioorthogonal enzymes for protein labelling. We will also look to develop partnerships with medical research organisations to develop diagnostic tools using our technology. Finally the PDRA who is trained during this project will develop key interdisciplinary skills, which will be extremely valuable to future industrial employers. Dr Wong will also get is first experience as co-I and he will gain valuable experience in project management and supervision. WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT: We will actively seek to communicate our findings to the wider community through scientific meetings and scholarly publications (We consistently publish in top journals JACS, PNAS, Angew. and Nature Chem. Biol ). Where appropriate to ensure that industrial and other future partners benefit from this work, we will secure intellectual property rights for all new inventions we discover. To this end, we will work closely with University KT staff and when possible we will seek follow-on funding from BBSRC and elsewhere. We have also enlisted project partners in a biotechnology consultancy (Dr. Farid Khan, see Impact Plan for further details) to enable us to establish links with potential partners in the biotechnology and pharmaceutical industries that would offer an avenue to exploit our research. We will also work closely with MIMIT, an organisation from the Univ of Manchester and Manchester NHS and Primary Care Trusts facilitating collaborations between clinicians, scientists, engineers and industry to develop innovative technology for patient benefit. Through MIMIT we will seek further follow-on funding to establish collaborations with medical community to develop protein (antibody) arrays as diagnostic tools. We will work closely with the university press offices in Manchester to ensure our work is disseminated to the general public.