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

Jason R. Waggoner, Jamie Huffman, Brian N. Griffith, Larry Jones, James E. Mahaney

Research output: Contribution to journalArticle

15 Citations (Scopus)

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

Fingerprint

Calcium-Transporting ATPases
Insects
phospholamban
Assays
Proteins
Muscle Relaxation
Baculoviridae
Phosphoproteins
Purification
Fluorescent Antibody Technique
Adenosine Triphosphatases
Muscle
Spectrum Analysis
Myocardium
Cytoplasm
Western Blotting
Spectroscopy

Keywords

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

ASJC Scopus subject areas

  • Biochemistry

Cite this

Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells. / Waggoner, Jason R.; Huffman, Jamie; Griffith, Brian N.; Jones, Larry; Mahaney, James E.

In: Protein Expression and Purification, Vol. 34, No. 1, 03.2004, p. 56-67.

Research output: Contribution to journalArticle

Waggoner, Jason R. ; Huffman, Jamie ; Griffith, Brian N. ; Jones, Larry ; Mahaney, James E. / Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells. In: Protein Expression and Purification. 2004 ; Vol. 34, No. 1. pp. 56-67.
@article{21b06f6cf087402cacb48313005299c2,
title = "Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells",
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.",
keywords = "Baculovirus, Ca-ATPase, High-Five cells, Phospholamban",
author = "Waggoner, {Jason R.} and Jamie Huffman and Griffith, {Brian N.} and Larry Jones and Mahaney, {James E.}",
year = "2004",
month = "3",
doi = "10.1016/j.pep.2003.11.005",
language = "English",
volume = "34",
pages = "56--67",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Improved expression and characterization of Ca2+-ATPase and phospholamban in High-Five cells

AU - Waggoner, Jason R.

AU - Huffman, Jamie

AU - Griffith, Brian N.

AU - Jones, Larry

AU - Mahaney, James E.

PY - 2004/3

Y1 - 2004/3

N2 - 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.

AB - 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.

KW - Baculovirus

KW - Ca-ATPase

KW - High-Five cells

KW - Phospholamban

UR - http://www.scopus.com/inward/record.url?scp=1442289363&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1442289363&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2003.11.005

DO - 10.1016/j.pep.2003.11.005

M3 - Article

VL - 34

SP - 56

EP - 67

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -