Recombinant histatins: functional domain duplication enhances candidacidal activity

Area of Science:

• Microbiology
• Biochemistry
• Protein Engineering

Background:

• Histatin 3 (Hst3) is a histidine-rich antimicrobial protein found in human saliva.
• The functional domain of Hst3 responsible for killing Candida albicans is located in its middle portion (amino acids 13-24).

Purpose of the Study:

• To establish a bacterial expression system for producing recombinant Hst3 (re-Hst3) and its variants.
• To investigate the structure-function relationship of Hst3 by creating and testing a variant with a repeated functional domain (re-Hst3rep).

Main Methods:

• Utilized PCR and splice overlap extension to create the re-Hst3rep variant.
• Employed the pRSET bacterial expression system for producing chimeric fusion proteins.
• Isolated and purified proteins using affinity chromatography and reverse-phase high-performance liquid chromatography.
• Assessed candidacidal activity using a Candida albicans killing assay.

Main Results:

• Recombinant Hst3 (re-Hst3) and the variant re-Hst3rep were successfully produced and purified.
• The variant re-Hst3rep exhibited significantly enhanced candidacidal activity compared to native Hst3 and re-Hst3.
• LD50 values for 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.

Conclusions:

• A functional bacterial expression system for producing biologically active Hst3 and its variants has been demonstrated.
• Repeating the functional domain of Hst3 significantly enhances its candidacidal activity.
• This study provides insights into Hst3 structure-function relationships and potential for developing enhanced antimicrobial agents.