Effects of calcium on recombinant bovine chromogranin A.
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(r) 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 Ca2(+)-dependent from 5 to 40 microM.
Angeletti
R H
RH
Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461.
Ali
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G
Shen
N
N
Gee
P
P
Nieves
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D053379:Chromogranin A; D002864:Chromogranins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002118:Calcium