Pilot Access for New Structural Biology Technologies through Instruct-ULTRA

 

Submit new proposal for this call

 

We invite applications from users who have a pre-existing research project which would benefit from one of the new techniques. The Open Call will involve a research visit to the host laboratory, with the user bringing their own sample in an appropriate form.

The purpose of this call is to test out the new technology and the user experience and your visit will help the final stages of technology refinement.

Details of each technology are given below.


Transient Gene Expression test for integral membrane proteins (TGE-IMP)

The expression of suitable constructs for the production of Integral Membrane Proteins (IMP) is analyzed by Transient Plasmid-Based Gene Expression in High-Five insect cells in either small scale (40 ml) or high throughput (40 samples in 1-2 ml scale) (Bleckmann et al. PlosOne 2019). The potential DNA sequence for the IMP and its variants will be cloned into an optimized expression vector pOpIE2-C6-mCherry as an in-frame C-terminal fusion to mCherry. The cloned DNA is purified and transfected in High-Five cells in 40 ml shake flasks or in the Biolector automated microfermentation system (40 constructs 1-2 ml). The expression of the IMP fusion protein is monitored for 72 hours by measuring the fluorescence of mCherry. The best expression construct is selected and can be used for large scale up (Litre) production of transfected cells. The Biolector system allows for direct evaluation of the optimal time of harvest. The cell pellets will be isolated and can be send to the user for further analysis.

Pilot expression test:
The pilot expression test is offered for a selected group of potential users (1-2 targets) to show the suitability of the new TGE-IMP technology for the catalogue of Instruct-ERIC services. The candidate should describe (a) the relevance of the target IMP for structural and functional analysis, (b) the available data of prior expression analysis of the IMP, (c) the requested number of constructs, (d) scale of the analysis and (e) describe the required fusion tag for purification (Twinstrep-, His8-, Flag or other). In the pilot phase the construction of the backbone of the vector for cloning the IMP will be offered by the facility. In the service phase the user will be able to select the optimal fusion vector for expression from the catalogue.

Contact:
Joop van den Heuvel, joop.vandenheuvel@helmholtz-hzi.de 

Address:
Helmholtz Centre for Infection Research
Protein Sample Production Facility
Inhoffenstrasse 7
38124 Braunschweig
Germany

References:
Bleckmann et al. (2016) Biotechnol Bioeng 116
Bleckmann et al. (2019) PLoS ONE 14(6): e0217878 


Bioreactor for time-resolved in-cell NMR in Florence

This innovative technology allows performing in-cell NMR experiments on human cells for prolonged periods of time. The bioreactor ensures cell viability and metabolic stability through a continuous flow of fresh nutrients and removal of toxic by-products. Time-resolved in-cell NMR experiments can be recorded for up to 72 hours and can be applied to monitor biological events in real time involving protein structural changes, such as folding, maturation, cofactor binding, protein-ligand and protein-protein interactions. Proteins will be expressed and observed in HEK293T cells.

Requirements:
Protein of interest amenable to solution NMR; to assess sample suitability it is recommended to have preliminary NMR data (ideally, solution in-cell NMR data). Due to the challenging nature of the technique, requirements will be evaluated on a case-by-case basis. User should bring the gene of interest cloned in a suitable vector for mammalian expression and any reagent (cofactors/small molecules) needed to perform the proposed experiments.

Contact:
Francesca Morelli, fmorelli@cerm.unifi.it
Enrico Luchinat, eluchinat@cerm.unifi.it

Address:
Magnetic Resonance Center
University of Florence
via Luigi Sacconi 6
50019 Sesto Fiorentino (Florence)
Italy

In Cell NMR in Grenoble

Specific labelling of methyl groups for the study of large protein in eukaryotic cells using NMR.

Requirement: expression of the target protein in Sf9 or HEK cells with a minimal yield of 20 mg/L of culture.

Applicants should be doctoral or postdoctoral researchers from an academic or industry setting with previous experience in isotopic labelling of protein produced in eukaryotic cells.

Contact: Jerome Boisbouvier, jerome.boisbouvier@ibs.fr

Address:
Institut de Biologie Structurale
71 Avenue des Martyrs
CS 10090
38044 GRENOBLE CEDEX 9
FRANCE

Phone: (+33) 4 57 42 86 71

 

Endogenous protein tagging for integrative structural biology applications

Molecular complexes of interacting proteins govern virtually all biological processes. In collaboration with the TacGene platform*, Strasbourg proposes the generation of modified cell lines expressing an affinity tagged protein and large scale cultures to facilitate the isolation of associated protein complexes for proteomic or structural biology applications. Although endogenous protein tagging can be used to identity new protein complexes, projects which focus on characterized assemblies (the expression of the target protein in the chosen cell line should be documented and antibodies directed against several subunits of the complex are needed) will be favoured.

*The TacGene platform (see https://biophysique.mnhn.fr/site/TACGENE-en) hosted by U1154-UMR7196 offers access advanced solutions and expertise on the various issues of genome editing (knock-out, knock-in ...) currently available.

Contact: Arnaud Poterszman, Arnaud.Poterszman@igbmc.fr

Address: CBI/IGBMC, 1 rue Laurent Fries 67 404 Illkirch cedex 

Call Submission Guidelines

Eligibility

Applicants can be pre-doctoral scientists (PhD students), or postdoctoral researchers from an academic or industry setting.

Applications should focus on the benefit to the applicant’s research. 

To apply for the Open Call, you will first need to set up a user account on ARIA. When you click to submit a new proposal for this call, you will be automatically directed to a page where you can set up or log in to an ARIA account.

Terms

The Open Call will fund machine access and consumables, paid to the host laboratory. A cap is set on €3000 for consumables.

Successful applicants will be expected to fund their own travel and accommodation for the laboratory visit.

Within 3 months of the completion of the internship platform access, applicants must give visit feedback and submit a short report about their research and visit experience. 

All proposals submitted by industry will be kept confidential. Any questions about legal agreements can be addressed to the host research centre.

Call Closes:
02-Feb-2020 23:55 CET

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