PSI Protein Production and HTX

The PSI Protein Production Facility

The PSI Protein Production Facility offers membrane protein expression and purification in mammalian cells for structural biology. The facility offers access to sophisticated equipment and technologies and provides advice and technical assistance in sample preparation. The facility provides small-scale expression and analysis of constructs, upscaled mammalian cell expression, purification and analysis in various detergents.

  1. The user will generate their constructs in their home laboratory or in collaboration with the PSI Protein Production Facility prior to the visit.
  2. Appropriate constructs fused to GFP/YFP will be selected using small-scale expression in HEK293 cells followed by solubilisation in DDM and detection of expression level using fluorescence-detection size-exclusionchromatography.
  3. Upscale transient expression in HEK293 cells using roller bottles or equivalent.
  4. Membrane preparation and solubilisation in DDM.
  5. Purification of target membrane proteins by IMAC in the presence of various detergents.
  6. Analysis of activity (user) and/or stability of purified membrane proteins by SEC and biophysical methods (PSI).

Users will typically stay approximately two to three weeks.

Users are offered the opportunity to set up crystallisation trials through the BioStruct-X-funded PSI Crystallisation Facility. Charges for using this facility will be handled separately and will require a new proposal submission through the BioStruct-X hub.

The PSI Crystallisation Facility

The PSI Crystallisation Facility provides state-of-the-art high-throughput crystallisation services for structural biologists at PSI, as well as European users through the BioStruct-X programme.
Services provided by the facility:
The PSI Crystallisation Facility provides a full crystallisation service. We offer access to sophisticated equipment and technologies, but users may also send us their samples. We also offer advice and technical assistance in sample preparation. The facility will provide screen preparation, automated plate setup, automated image acquisition and analysis, and, if desired, optimisation design. The facility combines technological advances to automate every step along the crystallisation process. We offer “standard” (Phoenix and Hamilton robots) and liquid cubic phase (Mosquito) crystallisation. Furthermore, we provide in-plate screening at the SLS.

In summary, we offer:

  • Personal consultation, guidance, and technical assistance to researchers on macromolecular crystal growth
  • Automated and/or manual setup of crystallisation conditions using a minimal amount of protein sample
  • Sample analysis and crystal identification
  • Optimisation of crystallisation conditions using 96 or 24-well formats
  • Storage of plates in temperature-controlled incubators

Analysis

After diffracting crystals are obtained, computers and software are available for the computation-intensive steps of data processing, reduction, and analysis, as well as graphics-intensive model-building and refinement.

 
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BioStruct-X is part of the Capacities Specific Programme Research Infrastructures, Project Number 283570
BioStruct-X contact: biostructx(at)embl-hamburg.de, I. Custic, ph: +49 - 40 89902 124, fax: +49 - 40 89902 149

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