Final Report Summary - ENEFP (European Network on Directed Evolution of Functional Proteins)
This project focused on using directed evolution for generating enzyme catalysts. The network implemented a training programme to advance conceptual and technological approaches for harnessing the forces of Darwinian evolution in the laboratory to
(i) Screen large and diverse genome libraries for proteins with new and useful functions,
(ii) Optimize existing proteins for applications in medicine, biotechnology and cell biology and
(iii) Provide a better understanding of how existing enzymes evolved and study enzyme mechanisms in general.
The Network brings together academic and industrial groups with diverse and complementary skills: the development of a variety of innovative biotechnology tools for the generation and exploitation of large genomic and man-made libraries, expertise in mechanistic enzymology and a technology platform including phage display, in vitro compartmentalization, ribosome display, selective protein labelling and high-throughput screening in microfluidic microdroplets.
In pursuing these goals we have formed lasting partnerships between the academic laboratories and the three companies that were involved in collaborative projects with other network groups and in hosting young researchers. Specific projects include:
• MedImmune (http://www.medimmune.com/) has an advanced proprietary technology for rapidly isolating human monoclonal antibodies using Phage Display and Ribosome Display systems, including extensive phage antibody libraries, as the source for a portfolio of antibody-based drugs. The network allowed MEDI to start a discovery effort in catalytic biologicals, using enzymatic catalysts in addition to conventional binding strategies. The network project (with Cambridge University) was awarded a prize for the best collaborative project with an academic lab in an internal competition.
• BRAIN AG (http://www.brain-biotech.de) offers integrated contract research services and characterized enzyme libraries to customers in the fine chemical, pharmaceutical, consumer care and food/feed industries. Its core expertise lies in the molecular exploitation of microbial resources and collaborations with the universities of Greifswald and Cambridge were pursued in this project.
• Covalys AG: this start-up company in the field of protein science was set up by a network partner (Prof. Johnsson at Lausanne) and focuses on protein immobilization and labeling techniques used for proteomics and drug discovery by exploiting a technology for highly specific and stable protein labeling and immobilization, the so-called SNAP-tag. The SNAP-tag that was used in projects at ETH Lausanne, Cambridge and MEDImmune. During the network period Covalys was bought by New England Biolabs, demonstrating the value of biotech inventions by a successful exist strategy – SNAP-tag products are now available at
http://www.neb.com/nebecomm/tech_reference/gene_expression_cellular_analysis/SNAP_tag_technologies.asp
The collaborative and individual projects pursued in this network include the following:
• Evolution of a penicillin binding protein into a beta-lactamase, highlighting the fundamental challenge of converting a binding into a catalytic scaffold
• Development of therapeutic proteases (MEDImmune/Cambridge)
• Development of a new protein display system for directed evolution (MedImmune/UK, Cambridge/UK, Lausanne/CH)
• Evolution of cytosolic sulfotransferases (Bersheva, IL)
• Study of Catalytically promiscuous enzymes as starting points for directed evolution (Cambridge, UK)
• Generating a ratiometric fluorescent biosensor based on Förster resonance energy transfer (FRET) for the neurotransmitter g-aminobutyric acid (Lausanne, CH)
• Optimising a lipase for heterologous expression (Brain AG, D)
• The discovery of deamination enzymes (ammonia lyases) to synthesise non-natural amino acids (Groningen, NL)
• Evolution of esterases for higher stereoselectivity (Greifswald, D and Brain AG)
• Development of high-throughput assays in microdroplets (Strasbourg/F, Cambridge/UK, Brain AG, MedImmune)
The training programme for young researchers included:
• Local training formats (including complementary skills, hands-on training, experimental techniques)
• A summer school on protein engineering by rational and irrational methods
• Network meetings with talks by all participating groups and invited guest speakers
• Technical workshops, specifically on droplet-based directed evolution methods
• Lectures and workshops on IP protection, entrepreneurship and the industry/academia interface
• A three-day conference (http://biotech.uni-greifswald.de/pdb2012.html) in August 2012 at Greifswald that attracted more than 150 external participants: in addition to highlighting the network members’ research international guest speakers were invited. The conference attracted about 20% attendees from industry and helped to spread the approaches developed in the network into the private sector.
(i) Screen large and diverse genome libraries for proteins with new and useful functions,
(ii) Optimize existing proteins for applications in medicine, biotechnology and cell biology and
(iii) Provide a better understanding of how existing enzymes evolved and study enzyme mechanisms in general.
The Network brings together academic and industrial groups with diverse and complementary skills: the development of a variety of innovative biotechnology tools for the generation and exploitation of large genomic and man-made libraries, expertise in mechanistic enzymology and a technology platform including phage display, in vitro compartmentalization, ribosome display, selective protein labelling and high-throughput screening in microfluidic microdroplets.
In pursuing these goals we have formed lasting partnerships between the academic laboratories and the three companies that were involved in collaborative projects with other network groups and in hosting young researchers. Specific projects include:
• MedImmune (http://www.medimmune.com/) has an advanced proprietary technology for rapidly isolating human monoclonal antibodies using Phage Display and Ribosome Display systems, including extensive phage antibody libraries, as the source for a portfolio of antibody-based drugs. The network allowed MEDI to start a discovery effort in catalytic biologicals, using enzymatic catalysts in addition to conventional binding strategies. The network project (with Cambridge University) was awarded a prize for the best collaborative project with an academic lab in an internal competition.
• BRAIN AG (http://www.brain-biotech.de) offers integrated contract research services and characterized enzyme libraries to customers in the fine chemical, pharmaceutical, consumer care and food/feed industries. Its core expertise lies in the molecular exploitation of microbial resources and collaborations with the universities of Greifswald and Cambridge were pursued in this project.
• Covalys AG: this start-up company in the field of protein science was set up by a network partner (Prof. Johnsson at Lausanne) and focuses on protein immobilization and labeling techniques used for proteomics and drug discovery by exploiting a technology for highly specific and stable protein labeling and immobilization, the so-called SNAP-tag. The SNAP-tag that was used in projects at ETH Lausanne, Cambridge and MEDImmune. During the network period Covalys was bought by New England Biolabs, demonstrating the value of biotech inventions by a successful exist strategy – SNAP-tag products are now available at
http://www.neb.com/nebecomm/tech_reference/gene_expression_cellular_analysis/SNAP_tag_technologies.asp
The collaborative and individual projects pursued in this network include the following:
• Evolution of a penicillin binding protein into a beta-lactamase, highlighting the fundamental challenge of converting a binding into a catalytic scaffold
• Development of therapeutic proteases (MEDImmune/Cambridge)
• Development of a new protein display system for directed evolution (MedImmune/UK, Cambridge/UK, Lausanne/CH)
• Evolution of cytosolic sulfotransferases (Bersheva, IL)
• Study of Catalytically promiscuous enzymes as starting points for directed evolution (Cambridge, UK)
• Generating a ratiometric fluorescent biosensor based on Förster resonance energy transfer (FRET) for the neurotransmitter g-aminobutyric acid (Lausanne, CH)
• Optimising a lipase for heterologous expression (Brain AG, D)
• The discovery of deamination enzymes (ammonia lyases) to synthesise non-natural amino acids (Groningen, NL)
• Evolution of esterases for higher stereoselectivity (Greifswald, D and Brain AG)
• Development of high-throughput assays in microdroplets (Strasbourg/F, Cambridge/UK, Brain AG, MedImmune)
The training programme for young researchers included:
• Local training formats (including complementary skills, hands-on training, experimental techniques)
• A summer school on protein engineering by rational and irrational methods
• Network meetings with talks by all participating groups and invited guest speakers
• Technical workshops, specifically on droplet-based directed evolution methods
• Lectures and workshops on IP protection, entrepreneurship and the industry/academia interface
• A three-day conference (http://biotech.uni-greifswald.de/pdb2012.html) in August 2012 at Greifswald that attracted more than 150 external participants: in addition to highlighting the network members’ research international guest speakers were invited. The conference attracted about 20% attendees from industry and helped to spread the approaches developed in the network into the private sector.