Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs.
Saliva is a supersaturated solution with respect to hydroxyapatite, the main inorganic component of tooth enamel. Several acidic phosphoproteins are present in saliva which allow the supersaturated state to be maintained without random crystallization occurring. Statherin is the only salivary protein currently known to inhibit both the primary and secondary precipitation of hydroxyapatite in the supersaturated environment of saliva. To identify the residues of statherin that are necessary to control biomineralization, a recombinant form of human statherin was produced from Escherichia coli using a yeast intein fusion construct. The primary structure of the recombinant statherin was characterized by SDS-PAGE, N-terminus sequencing, MALDI mass spectrometry, and amino acid analysis and found to have the expected values relative to human-derived statherin. The secondary structure of the recombinant statherin was investigated by circular dichroism spectroscopy, which revealed the predominant presence of random coil in phosphate-buffered saline solution, with a higher propensity toward alpha helicity in 100% TFE. This increase in helicity in 100% TFE was also found in statherin that was synthesized by solid-phase synthesis. These results demonstrate that human statherin can be produced in a recombinant form which behaves comparably to the natural form.
Department of Bioengineering, University of Washington, Seattle, Washington 98195, USA.
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M
Gilbert
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PS
Stayton
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C018149:STATH protein, human; D012471:Salivary Proteins and Peptides