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.
Douglas
Jennifer L
JL
Department of Biochemistry, University of Alberta, Edmonton, Alta., Canada T6G 2H7.
Trieber
Catharine A
CA
Afara
Michael
M
Young
Howard S
HS
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D000252:Calcium-Transporting ATPases / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D011510:Proteolipids / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D009124:Muscle Proteins; D011510:Proteolipids; D011994:Recombinant Proteins; D000099211:Phospholamban; C077258:sarcolipin