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 t reatment of the intestinal tissue followed by acetic acid extraction, a cap ture with alginic acid, NaCl precipitation of other proteins, and a concent ration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities o f pure calbindin. In the second method, an additional ion exchange HPLC ste p was introduced, followed by a reverse-phase HPLC resulting in 100 milligr am-scale preparations of homogeneous calbindin in a 56% yield from the Ambe rlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrome try corresponding to N-acetylated porcine calbindin. The isolated apocalbin din was fully reconstituted with 2 molar equivalents of Ca2+ and the protei n displayed UV and fluorescence spectra characteristic of those of native c albindin D9k. (C) 1999 Academic Press.