New Application Note Reveals Wyatt Technology’s MALS Instrument Achieves Ultimate Purification of the Rab11-FIP2 Complex

A recent application by the School of Biochemistry and Immunology of the Trinity College in Dublin, Ireland demonstrates that Wyatt Technology’s miniDAWN™ Multi-Angle Light Scattering (MALS) instrument measures the finest purification of the Rab11-FIP2 complex. The findings of this analysis are detailed in a new, free-of-charge application note available from Wyatt, the world leader in absolute macromolecular characterization products.

The small GTPase Rab11 regulates the recycling of endosomes to the plasma membrane via interactions with the Rab11 Family of Interacting Proteins (FIPs). FIPs contain a highly conserved Rab-Binding Domain (RBD) at their C-termini. The overall structure is a hetero-tetramer with dyad symmetry, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 forms a central α-helical coiled-coil, with both helices contributing to the Rab11 binding patch on equivalent and opposite sides of the homodimer. The solution state of FIP2 needs to be characterized alone in order to confirm that Rab11 binds to a preformed complex of the FIP2 homodimer.

For this particular application, the constructs FIP2 were co-transformed into BL21 cells and over-expressed in 2xYT media supplemented with 100 mg/ml ampicillin and 30 mg/ml kanamycin at 37˚C. Following induction, cells were grown for a further 3h, harvested by centrifugation and stored at -20˚C. Frozen pellets were re-suspended in Maltose-Binding Protein (MBP) extraction buffer and sonicated at room temperature.

Cell lysates were centrifuged at 20,000g to remove cell debris and the resultant supernatant applied to an amylose resin. After extensive washing with MBP extraction buffer, bound protein was eluted with MBP elution buffer. Eluted protein was dialyzed overnight against 10mM Tris, 25mM NaCl in the presence of rTEV protease. Cleaved protein was loaded onto an ion-exchange column and a salt gradient was applied over a 20-fold excess column volume. The protein fractions corresponding to FIP2 were pooled and further purified on a superdex 200 16/60 column equilibrated in column buffer. The eluant was coupled to a miniDAWN and an Optilab RI.

The results of the analysis reveal that FIP2 is indeed a dimer in solution. Since Rab11 in solution is a monomer, the light scattering data suggest that FIP2 forms homodimers and recruits two molecules of Rab11 during trafficking of Rab11-positive endosomes.

For more information on Wyatt Technology’s miniDAWN™ MALS instrument or to obtain a copy of this application note, please visit the article webpage or click 'request info'.