Mammalian cell expression tools

Work Package 9

Mammalian Cell Toolbox for Structural Biology

Mammalian cell expression in HEK293 and CHO cells is well-proven in structural biology for generating folded, soluble forms of the most challenging proteins, frequently with native post-translational modifications, at a yield sufficient for down-stream crystallisation and other structural biology methods.

For certain target types, including membrane proteins and cell surface receptors, it can often be the only way to produce the material. However, the procedures are long and complex in comparison with simpler bacterial overexpression systems such as E. coli and this prevents its wider adoption as a tool in structural biology, thus limiting the range of projects upon which labs are prepared to work.

In WP9 we present developments that, during the first half of the BioStruct-X project, will generate a “mammalian cell expression toolbox for structural biology”. This will i) expand the repertoire of mammalian cell expression tools for expressing the most difficult targets for crystallisation trials and associated functional studies, and ii) simplify standard multi-step procedures through use of a large, specialised robotic system. The outputs will be a suite of tested protocols and materials (strains, vectors) that will be available to users in the protein production TNA WP5. Ultimately, this JRA will enhance the protein production TNA activities of WP5 and will thus lead directly to samples from some of the most challenging structural biology projects in Europe that will then use the complementary beamline-oriented applications of SAXS, MX and XI.

The key activities are:

  1. The development of library scale screening methods to optimise constructs, developing further the ESPRIT technology of EMBL Grenoble. This should enable expression of targets that comprise poorly annotated domains and which present folding problems in E. coli;
  2. Developing a baculovirus system capable of expressing protein complexes in both insect and mammalian cells. This will make overexpression and functional studies possible using the same virus construct;
  3. Developing new protocols for the TAP Select robot to increase the throughput of HEK293 expression experiments where the aim is to ease user access by replacing many of the tedious manual steps;
  4. Setting up new vector systems to control glycosylation levels of cell-surface proteins that should aid the crystallisation of receptors and other membrane proteins.

 

 
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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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