Expressing and purifying membrane transport proteins in high yield

Calvin C. Hale, Chananada K. Hill, Elmer M. Price, Julie B C Bossuyt

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni 2+ affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.

Original languageEnglish (US)
Pages (from-to)233-243
Number of pages11
JournalJournal of Biochemical and Biophysical Methods
Volume50
Issue number2-3
DOIs
StatePublished - Jan 4 2002
Externally publishedYes

Fingerprint

Membrane Transport Proteins
Larva
Recombinant Proteins
Purification
Polyacrylamide Gel Electrophoresis
Proteins
Sodium-Calcium Exchanger
Sodium Cholate
Affinity chromatography
Affinity Chromatography
Soybeans
Washing
Structural analysis
Detergents
Conformations
Phospholipids
Membrane Proteins
Western Blotting
Membranes
sodium-calcium exchanger 1

Keywords

  • Expression
  • Membrane protein
  • Transport

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

Expressing and purifying membrane transport proteins in high yield. / Hale, Calvin C.; Hill, Chananada K.; Price, Elmer M.; Bossuyt, Julie B C.

In: Journal of Biochemical and Biophysical Methods, Vol. 50, No. 2-3, 04.01.2002, p. 233-243.

Research output: Contribution to journalArticle

Hale, Calvin C. ; Hill, Chananada K. ; Price, Elmer M. ; Bossuyt, Julie B C. / Expressing and purifying membrane transport proteins in high yield. In: Journal of Biochemical and Biophysical Methods. 2002 ; Vol. 50, No. 2-3. pp. 233-243.
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