Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies

Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca(2+)-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca(2+)-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca(2+)-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca(2+)-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies.     

Nematode Memory: Now,Where Was I?

The nematode Caenorhabditis elegans is able to use tastes, smells and temperature to locate food. New data show that worms can also detect the level of oxygen in the environment and migrate towards an oxygen level associated with food.