The production and purification of recombinant proteins is a key step in the development or validation of antiviral molecules. The team "Viral replicases: Structure, Function and Drug Design" has developed extensive expertise in the field of production of recombinant viral proteins. This expertise was successfully used in several European projects of Genomics and Structural Virology (SPINE, VIZIER http://www.vizier-europe.org, SILVER http://www.silver-europe.com) in order to unravel viral replication mechanisms and increase the portfolio of potential antiviral targets.
Viral replication proteins are typically large multi-domain proteins difficult to produce in their full-length form. Our approach is to cut the polyproteins into functional modules that can be easily produced in a prokaryotic heterologous system (E. coli). This crucial step requires detailed analysis of viral sequences through the prism of the sequence conservation and the prediction of structured regions. We have developed a series of tools dedicated to this activity: a data base of viral sequences VaZyMolO (http://www.vazymolo.org) and the meta-server for the prediction of protein disorder MEDOR (http://www.vazymolo.org/MeDor/).
After having defined the limits of the protein domains the corresponding sequences are cloned into a series of bacterial expression vectors. Our projects led us to give preference to bacterial expression at a large scale over more costly eukaryote or in vitro expression systems. We have developed a panel of methods for optimization of bacterial protein expression. They are based on (i) screening of bacterial culture conditions (ii) search for "solubilizing" sequences (fused to the N part -terminus of the sequence of interest), (iii) optimization of non-coding sequences, (iv) renaturation of proteins from inclusion bodies (v) co-expression of heterologous proteins for the production of protein complexes.
In that way we have assembled a collection of expression vectors for viral replication proteins as well as of the corresponding proteins. Part of this collection has been made available to the scientific community within the European Research Infrastructure EVA (http://www.european-virus-archive.com).