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Effects of calcium on recombinant bovine chromogranin A
Author(s) -
Angeletti Ruth Hogue,
Ali Ghazala,
Shen Ning,
Gee Patricia,
Nieves Edward
Publication year - 1992
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560011208
Subject(s) - edman degradation , chromogranin a , chemistry , recombinant dna , biochemistry , size exclusion chromatography , gel electrophoresis , sodium dodecyl sulfate , circular dichroism , calcium , peptide sequence , escherichia coli , chromatography , microbiology and biotechnology , biology , enzyme , immunohistochemistry , organic chemistry , immunology , gene
Bovine chromogranin A, the acidic calcium‐binding protein characteristic of endocrine secretory vesicles, has been expressed in Escherichia coli using the pET3a vector system under T7 polymerase control. The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion‐exchange, and reversed‐phase chromatography. The purified recombinant chromogranin A has an apparent M T of ca. 72,000 by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, in spite of its 432‐amino acid polypeptide chain, consistent with observations on natural chromogranin A. The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence‐specific antibodies. Analysis by circular dichroism spectroscopy shows pH‐ and concentration‐dependent spectra. The spectra are Ca 2+ ‐dependent from 5 to 40 μM.