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

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)56-67
Number of pages12
JournalProtein Expression and Purification
Volume34
Issue number1
DOIs
StatePublished - Mar 2004

    Fingerprint

Keywords

  • Baculovirus
  • Ca-ATPase
  • High-Five cells
  • Phospholamban

ASJC Scopus subject areas

  • Biochemistry

Cite this