Histatin 1 is a histidine-rich phosphoprotein present in human parotid
saliva that possesses candidacidal activity and functions in minerali
zation by adsorbing to hydroxyapatite. The objective of the present st
udy was to develop a system for recombinant production of histatin 1 a
nd to examine the role of phosphorylation in the functional activities
of this molecule. Native histatin 1 (containing a phosphoserine at re
sidue 2) was purified from parotid saliva, whereas a bacterial express
ion system was used to produce a recombinant form of histatin 1 (re-Hs
t1) that lacked phosphorylated serine. Histatin 1 cDNA was inserted in
to the vector pGEX-3X, which expresses foreign genes as soluble fusion
proteins attached to the carboxyl-terminus of glutathione S-transfera
se (GST). The GST/re-Hst1 fusion protein was isolated from cell lysate
s by affinity chromatography on glutathione (GSH)-Sepharose and digest
ed with cyanogen bromide to separate re-Hst1 from the GST fusion partn
er. The digest was subjected to reversed-phase high-performance liquid
chromatography on a C-18 column, and re-Hst1 was eluted as a well-def
ined peak. The yield of re-Hst1 was 4 mg/L of bacterial culture. Amino
-terminal sequencing and amino acid analysis confirmed the final produ
ct as re-Hst1. Sodium dodecyl sulfate polyacrylamide gel electrophores
is (SDS-PAGE) showed that native histatin 1 and re-Hst1 had the same a
pparent molecular weights, while cationic PAGE showed that re-Hst1 was
more basic. Phosphate analysis indicated 1 mol phosphate/mol of nativ
e histatin 1, while re-Hst1 lacked any detectable phosphate. Re-Hst1 d
emonstrated candidacidal activity comparable to that of native histati
n 1, but displayed substantially lower binding to hydroxyapatite. Thes
e results show that phosphorylation of histatin 1 at residue 2 contrib
utes significantly to its ability to bind to hydroxyapatite.