Large-scale HPLC purification of calbindin D9k from porcine intestine

Valentina Bonetto, Liina Kangur, Peep Palumaa, Viktor Mutt, Hans Jörnvall, Rannar Sillard

Research output: Contribution to journalArticle

Abstract

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 ± 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca2+ and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.

Original languageEnglish
Pages (from-to)387-391
Number of pages5
JournalProtein Expression and Purification
Volume17
Issue number3
DOIs
Publication statusPublished - Dec 1999

ASJC Scopus subject areas

  • Biochemistry

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