Recombinant histatins: functional domain duplication enhances candidacidal activity.
Histatin 3 (Hst3) is a 32-amino-acid (aa) His-rich protein with antimicrobial activity found in human salivary secretions. To explore further 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 Hst3 (aa 13-24) contains the functional domain responsible for killing Candida albicans. Using PCR and splice overlap extension, a Hst variant (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 purified fusion proteins were digested with CNBr and re-Hst were separated from their fusion partners by reverse-phase high-performance liquid chromatography. The activity of re-Hst3 and re-Hst3rep was compared to that of native Hst3 from human salivary secretions in the C. albicans killing assay. The LD50 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 native Hst3 or re-Hst3 and at even lower concentrations re-Hst3rep exhibited significant candidacidal activity while native Hst3 and re-Hst3 were inactive. These results demonstrate an expression system for production of biologically active functional Hst and Hst variants and shows that repetition of the functional domain of Hst3 enhances candidacidal activity.
Zuo
Y
Y
Department of Periodontology and Oral Biology, School of Graduate Dentistry, Boston University Medical Center, MA 02118, USA.
Xu
T
T
Troxler
R F
RF
Li
J
J
Driscoll
J
J
Oppenheim
F G
FG
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015202:Protein Engineering; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D000099053:Histidine-Rich Glycoprotein
D000935:Antifungal Agents; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012471:Salivary Proteins and Peptides; D000099053:Histidine-Rich Glycoprotein