| Sign In to gain access to subscriptions and/or personal tools. |
Functional Comparison of Native and Recombinant Human Salivary Histatin 1Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, Boston, Massachusetts 02118
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, Boston, Massachusetts 02118
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, Boston, Massachusetts 02118
Department of Biochemistry, School of Medicine, Boston University Medical Center, Boston, Massachusetts 02118
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, Boston, Massachusetts 02118, Department of Biochemistry, School of Medicine, Boston University Medical Center, Boston, Massachusetts 02118
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, Boston, Massachusetts 02118, Department of Biochemistry, School of Medicine, Boston University Medical Center, Boston, Massachusetts 02118 Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hstl) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hstl fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hstl from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hstl was eluted as a well-defined peak. The yield of re-Hstl was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hstl. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hstl had the same apparent molecular weights, while cationic PAGE showed that re-Hstl was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hstl lacked any detectable phosphate. Re-Hstl demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.
Key Words: histatin • pGEX expression system • glutathione-Sepharose • phosphoprotein • candidacidal • hydroxyapatite binding
Journal of Dental Research, Vol. 74, No. 12,
1837-1844 (1995) This article has been cited by other articles:
|
|
|||||||||||||||||||||||||||
 |
|
|
 |
|
Sign In to gain access to subscriptions and/or personal tools.
