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Grenoble (GRB) provides access to users of the novel ESPRIT library-based construct screening technology. This was developed in the Hart laboratory at EMBL to identify soluble constructs of “difficult-to-express” protein targets that resist the classical approach of bioinformatics and PCR cloning. It employs the principles of directed evolution whereby a diverse random library of DNA constructs is generated and screened to identify rare clones of interest (soluble expressers) [PMID: 16369090]. All unidirectional truncations of the target gene, both 5' and 3', are synthesized by exonuclease degradation to generate potential expression constructs. Additionally, a “scanning” version identifies internal domains. In each experiment, up to 28,000 individual constructs are assayed in parallel for yield and solubility using colony picking and liquid handling robotics.
Recent examples of success include expression of soluble domains from the influenza polymerase PB2 subunit [17310249, 18454157, 18769709], H. pylori CagA [19143840] and H. sapiens NF1 [19111619], several of which have resulted in X-ray or NMR structure determination.
Modality of access and work
Construction of the construct library is performed in the user’s laboratory over a period of 3 to 4 weeks under (part time). This phase is remotely supervised by experts via a shared online labbook and requires a good level of molecular biology experience on the part of the user (no beginners) and the purchase of some standard reagents (enzymes and sequencing). The user then visits Grenoble for the robotic screening phase of 3 weeks duration. At the end of the project, the user will have plasmid constructs encoding soluble, purifiable constructs of any domains identified.
Support offered
At GRB, the user will be supervised on a daily basis through the experimental robotic procedures and will be supplied with the high throughput consumables for the work. Accommodation will be organised and the costs covered.
Figure 1: Screening tens of thousands of expression constructs of a target gene. Constructs are made as a random library and printed on membranes for soluble expression analysis by hybridisation of fluorescent antibodies. |
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Users:
