Larry R. Jones

School of Medicine, Medicine, Cardiology

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Improved expression and characterization of Ca²⁺-ATPase and phospholamban in High-Five cells

Jason R. Waggoner; Jamie Huffman; Brian N. Griffith; Larry R. Jones; James E. Mahaney (Profiled Author: Larry R. Jones)

Protein Expression and Purification. 2004;34(1):56-67.

The Ca²⁺-ATPase accounts for the majority of Ca²⁺ removed from the cytoplasm during cardiac muscle relaxation. The Ca ²⁺-ATPase is regulated by phospholamban, a 52 amino acid phosphoprotein, which inhibits Ca²⁺-ATPase activity by decreasing the apparent affinity of the ATPase for Ca²⁺. To study the physical mechanism of Ca²⁺-ATPase regulation by phospholamban using spectroscopic and kinetic experiments, large amounts of both proteins are required. Therefore, we developed a Ca²⁺-ATPase and phospholamban preparation based on the baculovirus-insect cell expression system using High-Five insect cells to produce large amounts of microsomal vesicles that contain either Ca²⁺-ATPase expressed alone or Ca²⁺-ATPase co-expressed with phospholamban. The expressed proteins were characterized using immunofluorescence spectroscopy, Ca²⁺-ATPase activity assays, Ca²⁺ uptake and efflux assays, and Western blotting. Our purification method yields 140 mg of microsomal protein per liter of infection (1.7 × 10⁹ cells), and the Ca²⁺-ATPase and phospholamban account for 16 and 1.4%, respectively, of the total microsomal protein by weight, yielding a phospholamban:Ca²⁺-ATPase ratio of 1.6:1, similar to that observed in native cardiac SR vesicles. The enzymatic properties of the expressed Ca²⁺-ATPase are also similar to those observed in native cardiac SR vesicles, and when co-expressed with phospholamban, the Ca²⁺-ATPase is functionally coupled to phospholamban similar to that observed in cardiac SR vesicles. © 2003 Elsevier Inc. All rights reserved.

PMID: 14766300    

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