Coordinator: 

Dr. Yoav Peleg
The Israel Structural Proteomics Center (ISPC), Faculty of Biochemistry
The Weizmann Institute of Science
Rehovot, 76100 | Israel
phone: + 972-8-9343191 / 2374
mail: yoav.peleg@weizmann.ac.il
web: www.weizmann.ac.il/~wspc

Dr. Renaud Vincentelli
Head of HTP Recombinant Protein Expression, Purification and Interaction
Structural Biology Core Facility, AFMB 
F-13288 MARSEILLE
phone: +33 (0)491.82.55.83
mail: renaud.vincentelli@afmb.univ-mrs.fr
web: http://www.afmb.univ-mrs.fr/cloning-expression-in-bacteria/

Scientific advisors:

Adi Goldenzweig
PhD candidate, Fleishman lab
Department of Biological Chemistry
Weizmann Institute of Science
phone:  + 972-8-9344967
mail: adi.goldenzweig@weizmann.ac.il
web: http://www.weizmann.ac.il/Biological_Chemistry/scientist/Fleishman/

Sarel Fleishman
Department of Biological Chemistry
Weizmann Institute of Science
mail: sarel@weizmann.ac.il
web: http://www.fleishmanlab.org

We have initiated a benchmarking study of the PROSS algorithm developed by Adi Goldenzweig in the lab of Sarel Fleishman (http://www.fleishmanlab.org) that may be used to improve the thermal stability of target proteins-the algorithm and protein stability data are described in Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

The algorithm designs multiple mutations (typically >10) in a target protein to improve surface polarity, core packing and backbone rigidity, without changing the active site, thus maintaining the functional properties. As a prime example, Adi designed human acetylcholinesterase (AChE), an enzyme where the wild-type version expresses so poorly in bacteria, that its purification in a functional state is barely feasible. Five PROSS-designed mutants were tested experimentally and displayed 100-1,800 fold higher expression levels in E. coli. The highest ranking design, encoding 51 substitutions relative to the human wild-type protein, was expressed at levels of ~ 2 mg per litre bacterial culture and had ~ 20°C higher resistance to heat-inactivation (compared to wild-type AChE expressed in HEK293 cells). Remarkably, four of the five mutants had catalytic rates against the acetylcholine substrate that were roughly identical to the wild-type enzyme. In another study, published in PNAS (One-step design of a stable variant of the malaria invasion protein RH5 for use as a vaccine immunogen), the same algorithm was used to substantially increase expression levels and thermal tolerance of a bio-medically relevant binding protein. Over 400 queries have already been submitted to PROSS from labs all over the world, and responses from researchers with no computational background have been enthusiastic. Adi’s presentation describing the PROSS can be downloaded using the following link:

PROSS-Designing stable and highly expressed variants of “tough-nut” proteins in one shot

The rationale for a P4EU PROSS benchmarking is:
1) To evaluate the success-rate of the PROSS algorithm in a systematic fashion by collecting and tracking the results of the optimizations. Ideally, the results will be presented in a collaborative publication including all participants
2) To help overcome yield limitations in the production of “hard-to express” proteins or generate variants that are more resistant to high temperature or other stringent conditions.
3) To strengthen the scientific ties within the P4EU community and increase visibility

Pre-requisites for proteins to be included in the study are:
1) A known X-ray structure, or structure of homologue with at least 50% sequence identity, and access to a number (>25 with more than 30% sequence identity) of protein sequences from different species.
2) An assay to compare the functional activity of the PROSS-designed mutants relative to wild-type.
3) The protein must be a ‘soluble’ protein and NOT a membrane protein.

To submit your protein data to PROSS please visit: http://pross.weizmann.ac.il

As the outcome of the project (tentative date – end of July 2017) we expect to be able to deliver the following information:
1) Protein expression: strains, culture conditions.
2) Expression level of the PROSS-designed variants compared to wild-type.
3) Activity of the PROSS-designed variants relative to wild-type.
4) Tm or inactivation temperature of selected variants compared to wild-type.
A collaborative publication will focus on the evaluation of the PROSS algorithm based on a collection of results and should not interfere with any individual publications arising from the study.