RECOMBINANT HISTATINS - FUNCTIONAL DOMAIN DUPLICATION ENHANCES CANDIDACIDAL ACTIVITY

Citation
Y. Zuo et al., RECOMBINANT HISTATINS - FUNCTIONAL DOMAIN DUPLICATION ENHANCES CANDIDACIDAL ACTIVITY, Gene, 161(1), 1995, pp. 87-91
Citations number
19
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
03781119
Volume
161
Issue
1
Year of publication
1995
Pages
87 - 91
Database
ISI
SICI code
0378-1119(1995)161:1<87:RH-FDD>2.0.ZU;2-T
Abstract
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.