We have developed new purification methods for membrane proteins expressed in Escherichia coli (E. coli), that is based on a high-throughput detergent solubilization screen and IMAC protein purification.
In case there is no previous research done on a particular membrane protein, this screening procedure starts with wide variety of detergents across a range of concentrations. Here we present an approach to solubilize a human sigma1 receptor expressed as N-terminal MBP fusion (with signal sequence that directs it to the periplasm – plasmid pVP87K). This protein has been extensively studied but the structure has yet to be solved . To perform this screen in a 96 well plate, it is sufficient to start with 0.1 g of wet cell paste. After breaking the cells by sonication, the lysate is centrifuged at 16,000g to separate the insoluble and soluble fractions. The pellet (insoluble fraction) is then re-suspended and used in the high throughput detergent solubilization screens. The testing is performed at 4ºC overnight, and then the solubilized fraction is separated by centrifugation. We employ the BioRad stain free imager to estimate the efficiency of solubilization. This method can be semi- or fully automated, and is serving as a precursor for automated purification using the Maxwell 16 system.
Brian G. Fox (firstname.lastname@example.org)
Transmembrane Protein Center
Last edited:Wed 26 Sep 2012 - 6 years, 2 months ago