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WP 6: Experimental validation


WP6 will be responsible for the experimental validation of the predictions made by the SSP software. Additionally, WP6 will reflect on the user-friendliness of the SSP.


WP6 will experimentally validate mutation predictions made with the SSP software and with BIOP's 3DM systems. The primary aim of these experiments is validation of the in silico predictions to the extent that the software and protocols can be improved using the validation results. WP6 has as secondary aim the actual production of improved enzymes.

The partners in WP6 will aim at different aspects of experimental validation. Partner EMAUG will provide its extensive knowledge in protein engineering in general as support to all three experimental SMEs. ENZYM, INGEN, and LEAD will use the SSP to tailor-design the proteins, which are of key interests to them, hence partner ENZYM will concentrate on transaminases and Baeyer-Villiger-monooxygenases (BVMO); partner INGEN will focus on aminotransferases, amine oxidases, and carboxylic acid reductases; while partner LEAD will base its in-house ongoing drug-design related protein engineering efforts on SSP results and will provide feedback on the outcome of those experiments.

Validation planning

EMAUG will use the bioinformatics tools available from the SSP and from BIOP to further accelerate the development of enzymes for biocatalysis. This will be performed for enzymes from the ~/~ hydrolase fold super-family where they aim for the inter-conversion of enzyme activities, e.g. convert an epoxide hydrolase into synthetically useful dehalogenases. Novel 3DM-systems will be developed for different enzyme families to further expand the research in this area to enable the identification of further useful transaminases, transferases, BMVO, oxidases, and reductases with respect to substrate range, enantioselectivity, and stability.

Thus, in close cooperation with partners 1, 3, and 5 targets for biocatalysis will be identified for the different enzyme classes. Bioinformatics information will be used by partners 2, 4, and 6 to design, create, and analyze mutant libraries. The best hits thus identified experimentally in the laboratory will be biochemically characterized to confirm predictions and their applicability in biocatalysis. This will be performed in close collaboration between the academic partner 2 and the experimental SMEs to take advantage of the high-throughput screening facilities and the protein engineering knowledge at EMAUG.

Task 1

Enzymicals will use the protein engineering SSP to tailor-design its biocatalyst on emerging issues. The primary focus will be on activity, selectivity and/or stability of transaminases and Baeyer-Villiger monooxygenases. Secondary targets are adjustments of the substrate spectra of representative catalysts from these two classes. Predicted mutations are compared with results of other available tools and selected positions are transferred to rational or evolutionary protein design. The obtained experimental results will be used to validate and refine the prediction tools and helps to understand the enzyme mechanisms. Improved variants will be used for the company~s catalogue business and incorporated in the in-house biocatalytic toolbox for the production of fine-chemicals.

Task 2

Ingenza will use the software tools available from the SSP and from BIOP for two particular classes of experiments. First the focus will be on the activity, selectivity, and thermostability of amino-transferase and carboxylic acid reductase enzyme super-families. These experiments will mainly use the multiple sequence alignment based tools. BIOP 3DM systems will be used to generate the experimental designs. Partners 1, 3, and 6 will carefully analyse the quality of these predictions and partner 3 will use this as input to a round of improvement of the software (and perhaps the science) they use for the production of 3DM systems. It takes partner BIOP days till weeks to produce one 3DM system. This is too big an effort in terms of CPU usage to be made freely available, so that EMBL's HSSP system (that is nowadays maintained by partner CMBI) will be used on the SSP for the same multiple sequence based purposes. HSSP alignments can be produced much faster than 3DM systems but will be less accurate. Partners 1 and 3 will carefully analyse the quality of HSSP based predictions and will try to improve the HSSP alignment system further, if possible, and if improvements will not unreasonably increase the CPU time efforts. The expected limitations of the predictions will be clearly documented (in collaboration with partner 7).

Ingenza will (need to) stabilize a few of its target enzymes. For this purpose they will use both the multiple sequence alignment based stability prediction techniques that are part of BIOP's 3DM system, and they will use the 'classical' protein structure and energy calculation based WHAT IF methods that will be incorporated in the SSP. The experimental results will be carefully analysed and these analyses will be made available to the users of the SSP. The experimental results will obviously also be used to generate ideas for better algorithms, protocols, or parameterisations for the prediction of stabilizing mutations.

Task 3

LeadPharma performs protein engineering experiments in many of its drug-design related projects. LeadPharma will not perform specially designed experiments for the purpose of SSP validation, but they will rather use the SSP as a normal tool in their in-house experimental design, and they will, in close consultation with partner 7 (SAFAN) report back to the NewProt team all its experiences. LeadPharma will be responsible for the validation of the SSP in a pharmaceutical (drug design) context.


Prediction and validation need to iterate continuously. WP6 will therefore continuously communicate with WP2-5. It is envisaged that most exchange of NewProt staff between the partner's labs will be related to this iteration process.

  The NewProt project is funded by the European Commission within its FP7 Programme, under the thematic area KBBE-2011-5 with contract number 289350.