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Immunoaffinity purification of parathyroid hormone‐related protein from bovine milk and human keratinocyte‐conditioned medium
Author(s) -
Stewart Andrew F.,
Wu Terrence L.,
Insogna Karl L.,
Milstone Leonard M.,
Burtis William J.
Publication year - 1991
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650060313
Subject(s) - affinity chromatography , parathyroid hormone related protein , polyclonal antibodies , amino acid , parathyroid hormone , chemistry , peptide sequence , biochemistry , antibody , biology , microbiology and biotechnology , gene , calcium , immunology , enzyme , organic chemistry
Parathyroid hormone‐related proteins (PRHrP) are a novel family of proteins that appear to be responsible for humoral hypercalcemia of malignancy. Although PTHrP derived from human tumors have been purified and their N‐terminal amino acid sequence determined, and although the structure of the PTHrP gene and its alternatively spliced mRNA transcripts have been defined, the secretory and circulating form(s) of the protein are unknown. Purification of PTHrP in the past has been difficult, requiring multiple chromatographic steps and months or years to complete. To define naturally occurring PTHrP species we have developed a rapid and efficient immunoaffinity purification method. Bovine milk (250 ml) and human keratinocyte‐conditioned medium (3000 ml) were affinity purified using a 300 μl affinity‐purified polyclonal anti‐PTHrP‐(1–36) antibody column and a single RP‐HPLC step. Purification required only 7–10 days and yielded a 3–4% recovery. Quantities of PTHrP sufficient for silver‐stained SDS‐PAGE, Western analysis, and N‐terminal amino acid sequence were obtained. In contrast to conventional purification schemes, affinity purification of PTHrP is rapid and efficient and can be applied to biologic samples that contain PTHrP in low abundance. These methods can be applied to the purification and characterization of the as yet undefined secretory and circulating forms of PTHrP.