Histatin 3 (Hst3) is a 32-amino-acid (aa) His-rich protein with antimi
crobial activity found in human salivary secretions. To explore furthe
r the structure/function relationship of Hst, we utilized a bacterial
system for the efficient production of recombinant Hst3 (re-Hst3) and
Hst variants. Previously, we demonstrated that the middle portion of H
st3 (aa 13-24) contains the functional domain responsible for killing
Candida albicans. Using PCR and splice overlap extension, a Hst varian
t (re-Hst3rep) was made in which the functional domain was repeated in
tandem. Using the pRSET bacterial expression system, re-Hst3 and the
variant re-Hst3rep were produced as chimeric fusions and were isolated
from bacterial sonicates by affinity chromatography. Affinity purifie
d fusion proteins were digested with CNBr and re-Hst were separated fr
om their fusion partners by reverse-phase high-performance liquid chro
matography. The activity of re-Hst3 and re-Hst3rep was compared to tha
t of native Hst3 from human salivary secretions in the C. albicans kil
ling assay. The LD(50) values for candidacidal activity of native Hst3
, re-Hst3 and re-Hst3rep were 7.2, 6.8 and 4.1 nmol/ml, respectively.
At lower concentrations re-Hst3rep was five times more active than nat
ive Hst3 or re-Hst3 and at even lower concentrations re-Hst3rep exhibi
ted significant candidacidal activity while native Hst3 and re-Hst3 we
re inactive. These results demonstrate an expression system for produc
tion of biologically active functional Hst and Hst variants and shows
that repetition of the functional domain of Hst3 enhances candidacidal
activity.