Large-scale HPLC purification of calbindin D9k from porcine intestine.
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 Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.
Bonetto
V
V
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, S-171 77, Sweden.
Kangur
L
L
Palumaa
P
P
Mutt
V
V
Jörnvall
H
H
Sillard
R
R
D000818:Animals; D064026:Calbindins; D002118:Calcium / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D007422:Intestines / Q000737:chemistry*; D013058:Mass Spectrometry; D011489:Protein Denaturation; D012117:Resins, Synthetic / Q000737:chemistry; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D064026:Calbindins; D012117:Resins, Synthetic; D064030:S100 Calcium Binding Protein G; D012965:Sodium Chloride; C046797:amberlite; D002118:Calcium