Development and demonstration of a trinucleotide exchange method for the directed evolution of proteins
Lead Research Organisation:
CARDIFF UNIVERSITY
Department Name: School of Biosciences
Abstract
Every living thing contains thousands of different proteins that carry out most of the crucial jobs needed to maintain life. Proteins are synthesised as a linear sequence of amino acids, which then fold to form their functional 3D structures. All the information required for a protein to reach its functional shape is encoded in the linear sequence of amino acids, which in turn is encoded by the DNA sequence of the gene for that protein. The process of evolution involves changes to the amino acid sequence of a protein through changes to the gene. As a consequence, these changes can alter the properties of the protein and may translate into beneficial effects, allowing the organism to survive a particular environmental challenge. How changes to the amino acid sequence of a protein translate into changes in the properties of a protein is one of the most fundamental questions in biology. The advent of genetic engineering has enabled us to change the DNA sequence of genes at will and so change the nature of the linear sequence of amino acids in the protein. This has allowed us to understand how certain amino acids contribute towards the properties of a protein. It has also allowed us to modify the characteristics of proteins for use in unnatural environments such as for industrial applications. The 3D structure of a protein is highly complex and our understanding of it is still limited. Therefore our ability to predict how a particular designed mutation will affect the properties of the protein is also limited. Nature takes a different, less rational approach, by introducing mutations at random and selecting only those mutations that are of benefit. As nature has already been very successful in adapting proteins for a multitude of functions, the proposed research aims to copy the process of evolution in the laboratory by developing a method that can introduce mutations randomly into a gene in order to change the properties of a protein. We will attempt to modify the characteristics of a protein called TEM-1 beta-lactamase, one of the proteins responsible for resistance to antibiotics such as penicillin. The advent of bacterial resistance to the traditional antibiotics led to the development of new and improved penicillin-like antibiotics. Nature was quick to respond and variants of TEM-1 soon evolved to overcome the toxic effects of these new antibiotics. Using our new method, the project will investigate how and which mutations contribute to adapting TEM-1 to detoxify these new antibiotics, helping us to understand the natural process by which this occurs.
Technical Summary
The aim of this project is to develop and demonstrate a new and generally applicable method for creating the molecular diversity that is a prerequisite for the directed evolution of proteins. Directed evolution is a powerful and successful protein engineering strategy that is based on the principles of natural Darwinian evolution. It involves the creation of molecular diversity by the introduction of mutations at random positions within a gene followed by the selection of appropriate protein variants with new or improved characteristics. Although a variety of methods exist for generating molecular diversity at the genetic level, there are several limitations. These include error, codon and amplification biases, sampling of a restricted set of amino acids and the masking of beneficial mutations by deleterious ones. To overcome these limitations, a new method called trinucleotide exchange (TriNEx) is proposed. TriNEx involves the removal of a single trinucleotide sequence at random positions throughout a gene of interest followed by its replacement with a second (randomised or defined) trinucleotide sequence. This will allow the generation of new protein variants with one amino acid substituted for any other amino acid at random positions throughout the polypeptide. It is a novel extension of a transposon-based directed evolution method recently developed in my laboratory to sample sequence and conformational diversity due to amino acid deletions. To demonstrate the feasibility of the method, the TriNEx concept will be applied to our model system, TEM-1 beta-lactamase. TriNEx will be used to construct a library of gene variants with trinucleotide substitutions spread throughout the gene encoding TEM-1. To show that TriNEx can introduce mutations that influence the properties of a protein, TEM-1 variants that have improved hydrolytic activity towards two normally poor substrates for the wild-type enzyme, will be selected from the library.
Organisations
People |
ORCID iD |
Dafydd Jones (Principal Investigator) |
Publications
Rohamare S
(2015)
Cloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp. NCIM 5124)
in Process Biochemistry
Reddington SC
(2013)
Different photochemical events of a genetically encoded phenyl azide define and modulate GFP fluorescence.
in Angewandte Chemie (International ed. in English)
Reddington SC
(2012)
Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry.
in Chemical communications (Cambridge, England)
Reddington SC
(2015)
Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence.
in Chemical science
Reddington S
(2013)
Genetically encoding phenyl azide chemistry: new uses and ideas for classical biochemistry.
in Biochemical Society transactions
Morris JL
(2013)
Aryl azide photochemistry in defined protein environments.
in Organic letters
Jones DD
(2011)
Recombining low homology, functionally rich regions of bacterial subtilisins by combinatorial fragment exchange.
in PloS one
Jones DD
(2008)
The role of loop and beta-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex.
in The Biochemical journal
Jones DD
(2014)
Transposon-based approaches for generating novel molecular diversity during directed evolution.
in Methods in molecular biology (Clifton, N.J.)
Description | New Resources: A new mutagenesis method to allow the directed evolution of a protein by the process of trinucleotide exchange and trinucleotide deletion. Several new reagents have been developed including an engineered transposon for trinucleotide deletion, a novel DNA cassette for trinucleotide addition, a series of plasmids to accompany the mutagenesis procedure. IP: The underlying transposon-based method for trinucleotide exchange and related derivatives for creating novel protein variants (including domain insertion and insertion-deletion protein variants) has been incorporated into a patent (PCT/GB2006/000187). This patent is currently at national phase (e.g. European Patent Application No. 06703246.6) so that the patent has worldwide protection. Research Collaborations: A new interdisciplinary and synthetic biology-orientated collaboration with chemists. Commercialisation potential: The work formed the basis for a Cardiff University i-Solv team which attempted to find ways to exploit the technology for commercial means (http://www.cardiff.ac.uk/news/articles/protein-power-4323.html). Our team and idea won the competition. |
Exploitation Route | The trinucleotide exchange (TriNEx) method proposed in the project addressed the limitations of the current methodology in protein engineering, so adding a vital new and general technology to the directed evolution toolbox. It utilises straightforward and routine molecular biology procedures making it accessible to non-specialists. Furthermore, TriNEx will form part of a suite of methods based around the same basic components, which allow the sampling of mutations not previously accessible by existing directed evolution methods but routinely used by nature to adapt proteins. Apart from amino acid substitutions, the suite of methods allows the sampling of amino acid deletions to the insertion of amino acids or whole protein domains. A common set of methods that access a wide variety of mutations will be very attractive to a researcher who wishes to use directed evolution to engineer their protein. The model protein chosen for this study, TEM beta-lactamase, is the main enzyme responsible for bacterial resistance to antibiotics such as penicillin. As part of the project, mechanisms were discovered as to how mutations alter the substrate specificity of TEM-1. This work is of interest to researchers trying to understand the molecular mechanisms behind the emergence of antibiotic resistance. Furthermore, it demonstrated that TriNEx can be used to improve or expand a biocatalytic processes. |
Sectors | Education Pharmaceuticals and Medical Biotechnology |
URL | http://www.cardiff.ac.uk/news/articles/protein-power-4323.html |
Description | (i) The underlying transposon-based method for trinucleotide exchange and related derivatives for creating novel protein variants (including domain insertion and insertion-deletion protein variants) has been incorporated into a patent (PCT/GB2006/000187). This patent is currently at national phase (e.g. European Patent Application No. 06703246.6) so that the patent has worldwide protection. (ii) As part of the project, mechanisms were discovered as to how mutations alter the substrate specificity of TEM-1. This work is of interest to researchers trying to understand the molecular mechanisms behind the emergence of antibiotic resistance. Furthermore, it demonstrated that TriNEx can be used to improve or expand a biocatalytic processes. (iii) Both the PI and PDRA have taken part in the Cardiff University 'Open Day' whereby local schools and colleges visit Cardiff. We explained how understanding and engineering enzymes are important with regards to many different products used every day - from cheese to washing powder to pharmaceuticals. The PI has also taken part in other engagement activities using the results from the work to underlie the important of topics including GMOs and synthetic biology. |
First Year Of Impact | 2009 |
Sector | Chemicals,Education,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | BBSRC-facing studentship |
Amount | £90,000 (GBP) |
Organisation | Cardiff University |
Sector | Academic/University |
Country | United Kingdom |
Start | 09/2014 |
End | 10/2017 |
Description | Cardiff Synthetic Biology Initiative |
Amount | £45,108 (GBP) |
Organisation | SynbiCITE |
Sector | Academic/University |
Country | United Kingdom |
Start | 04/2014 |
End | 07/2015 |
Description | High-throughput engineering of proteins with a reprogrammed genetic code |
Amount | £314,170 (GBP) |
Funding ID | BB/H003746/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2009 |
End | 09/2012 |
Description | Incorporation of unnatural amino acids into proteins by directed evolution |
Amount | £38,395 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2008 |
End | 09/2009 |
Description | MRC DPFS |
Amount | £41,135 (GBP) |
Organisation | Cardiff University |
Sector | Academic/University |
Country | United Kingdom |
Start | 12/2009 |
End | 02/2011 |
Description | Sparking with Impact Award |
Amount | £16,965 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2014 |
End | 06/2014 |
Title | Transposon-directed evolution |
Description | I have developed a series of protein mutagenesis technologies based on work at Cardiff that has been patented (WO 2006077411). The patent uses an engineered transposon to introduce random but defined breaks into a target gene that undergoes further downstream manipulations to generate mutations not sampled by existing directed evolution approaches. These include, trinucleotide deletion, trinucleotide replacement, TAG scanning mutagenesis and domain insertion. |
Type Of Material | Biological samples |
Year Produced | 2008 |
Provided To Others? | Yes |
Impact | My research group has used the method to construct new and useful protein variants (see publication list) and has formed the basis for future grant funding. Other groups are also exploiting the method to generate useful protein variants. |
Description | High-throughput engineering of proteins: Sampling extended chemical diversity by combining directed evolution with an expanded genetic code. BB/H003746/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | Application of the trinucleotide exchange process to unnatural amino acid incorporation. |
Collaborator Contribution | Non-natural amino acid systems. |
Impact | A multi-disciplinary grant between School of Chemistry and Bioscience Papers (DOI): 10.1021/ol3028779 10.1039/c2cc31887c 10.1007/978-1-4939-1053-3_11 10.1042/BST20130094 10.1039/b904031e 10.1002/anie.201301490 |
Start Year | 2007 |
Description | High-throughput engineering of proteins: Sampling extended chemical diversity by combining directed evolution with an expanded genetic code. BB/H003746/1 |
Organisation | Cardiff University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Application of the trinucleotide exchange process to unnatural amino acid incorporation. |
Collaborator Contribution | Non-natural amino acid systems. |
Impact | A multi-disciplinary grant between School of Chemistry and Bioscience Papers (DOI): 10.1021/ol3028779 10.1039/c2cc31887c 10.1007/978-1-4939-1053-3_11 10.1042/BST20130094 10.1039/b904031e 10.1002/anie.201301490 |
Start Year | 2007 |
Description | Merck KGaA BBSRC DTG Quota Case |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | We introduced the general concept and the methodology by which to construct potentially useful protein sensors systems through the use of the domain insertion strategy. The project would be related to the construction of GFP-based sensing systems by which GFP fluorescence would be modulated by secondary events. |
Collaborator Contribution | They provided expertise in fluorescence and access to equipment at their German base in Darmstadt for fluorescence life-time measurement. |
Impact | The most useful output has been several papers and a trained PhD student (Arpino). Papers (DOIs): 10.1007/978-1-4939-1053-3_11 10.1039/b904031e 10.1021/ja301987h 10.1016/j.str.2014.03.014 10.1371/journal.pone.0047132 10.1107/S139900471401267X |
Start Year | 2007 |
Description | Merck KGaA BBSRC DTG Quota Case |
Organisation | Merck |
Country | Germany |
Sector | Private |
PI Contribution | We introduced the general concept and the methodology by which to construct potentially useful protein sensors systems through the use of the domain insertion strategy. The project would be related to the construction of GFP-based sensing systems by which GFP fluorescence would be modulated by secondary events. |
Collaborator Contribution | They provided expertise in fluorescence and access to equipment at their German base in Darmstadt for fluorescence life-time measurement. |
Impact | The most useful output has been several papers and a trained PhD student (Arpino). Papers (DOIs): 10.1007/978-1-4939-1053-3_11 10.1039/b904031e 10.1021/ja301987h 10.1016/j.str.2014.03.014 10.1371/journal.pone.0047132 10.1107/S139900471401267X |
Start Year | 2007 |
Description | Single protein molecule ET and nanotech. EP/J015318/1 |
Organisation | Cardiff University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | The generation of engineered proteins for use in protein-gold and protein-sp2 system interfacing. |
Collaborator Contribution | Single protein molecule measurement and analysis |
Impact | This is a multi-disciplinary project involving the School of Bioscience and Physics. Recent papers (DOI): 10.1039/c2cc34302a 10.1002/smll.201102416 10.1039/c2nr32131a 10.1021/nn2036818 10.1021/nl103334q |
Start Year | 2006 |
Description | Single protein molecule ET and nanotech. EP/J015318/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | The generation of engineered proteins for use in protein-gold and protein-sp2 system interfacing. |
Collaborator Contribution | Single protein molecule measurement and analysis |
Impact | This is a multi-disciplinary project involving the School of Bioscience and Physics. Recent papers (DOI): 10.1039/c2cc34302a 10.1002/smll.201102416 10.1039/c2nr32131a 10.1021/nn2036818 10.1021/nl103334q |
Start Year | 2006 |
Description | Understanding the Mechanism of Membrane Protein Insertion. BB/M000249/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Country | United Kingdom |
Sector | Public |
PI Contribution | On going and concerns the application of non-natural amino acid protein engineering to protein secretion complexes. |
Collaborator Contribution | Supply of membrane protein secretion systems. |
Impact | On going. |
Start Year | 2013 |
Description | Understanding the Mechanism of Membrane Protein Insertion. BB/M000249/1 |
Organisation | University of Bristol |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | On going and concerns the application of non-natural amino acid protein engineering to protein secretion complexes. |
Collaborator Contribution | Supply of membrane protein secretion systems. |
Impact | On going. |
Start Year | 2013 |
Title | POLYPEPTIDE MUTAGENESIS METHOD |
Description | There is provided a method for altering the amino acid sequence of a target polypeptide by altering a target DNA sequence which encodes that polypeptide, the method comprising the step of introducing a transposon into the target DNA sequence, in which the transposon comprises a first restriction enzyme recognition sequence towards each of its termini, the recognition sequence not being present in the remainder of the transposon, or in the target DNA sequence, or in a construct comprising the target DNA sequence, the first restriction enzyme recognition sequence being recognised by a first restriction enzyme which is an outside cutter and being positioned such that the first restriction enzyme has a DNA cleavage site positioned beyond the end of the terminus of the transposon. |
IP Reference | WO2006077411 |
Protection | Patent application published |
Year Protection Granted | 2006 |
Licensed | Yes |
Impact | The generation of new and useful fluorescent proteins. |
Description | Invited speaker at conferences |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Participants in your research and patient groups |
Results and Impact | I have talked at many different conferences presenting work related to the funded work. Making contacts and advertising the work supported by the grants. |
Year(s) Of Engagement Activity | 2006,2007,2008,2009,2010,2011,2012,2013 |
Description | School Visit (Wales) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | talk and discussion with students Informed students on current science |
Year(s) Of Engagement Activity | 2009 |
Description | Talks at Universities. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Participants in your research and patient groups |
Results and Impact | Talks to students, PhDs, postdocs and PIs in areas related to those associated with the grants. Collaborations. |
Year(s) Of Engagement Activity | 2006,2007,2008,2009,2010,2011,2012,2013 |