FRISBI

logo frisbi

FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY

Bacterial-expression.jpg

AFMB

BACTERIAL EXPRESSION

The cloning, expression and protein purification facility of the AFMB (IBiSA) is opened to customers since 2003 and is linked to the protein characterization and crystallography facilities of the lab.

We use the Gateway technology (Life Tech) in 96 well format but other cloning methods are possible. The cloning is done using a template supplied by the customer or a synthetic gene. The clones are sequenced and sent to the customers.

After cloning the gene of interest in one or several expression vectors, we optimize the level of soluble expression at analytical scale on a Tecan robot playing with various culture conditions and protein fusions. We do a 1-5 litres culture to purify the protein in suitable quantities for the customers only once we have found optimal conditions for the soluble expression.We and others have demonstrated the impact of the culture conditions and plasmid constructs on the level of expression and solubility. To be able to quickly screen for optimal conditions we have developped and published a high throughput expression screen protocol based on an automated dotblot. We are able to work on up to 1152 cultures/week with 1 ml culture in deep-well 96.

If we detect only the protein in inclusion bodies we use our own home made renaturation kit to try to refold the proteins and purify them in native conditions. We can also purify the proteins in a denatured form (to produce antibodies for instance).

 

For more details see: http://www.afmb.univ-mrs.fr/cloning-expression-in-bacteria