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.

Publications

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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