Journal Article

High-Yield Preparation of American Oyster Defensin (AOD) via a Small and Acidic Fusion Tag and Its Functional Characterization

Yang, Na, Wang, Jianhua, Li, Yuanyuan, Hao, Ya, Teng, Da, Mao, Ruoyu, Gu, Xinxi, Lu, Haiqiang, Zhao, Qingyi

2023

Overview

The marine peptide, American oyster defensin (AOD), is derived from Crassostrea virginica and exhibits a potent bactericidal effect. However, recombinant preparation has not been achieved due to the high charge and hydrophobicity. Although the traditional fusion tags such as Trx and SUMO shield the effects of target peptides on the host, their large molecular weight (12–20 kDa) leads to the yields lower than 20% of the fusion protein. In this study, a short and acidic fusion tag was employed with a compact structure of only 1 kDa. Following 72 h of induction in a 5 L fermenter, the supernatant exhibited a total protein concentration of 587 mg/L. The recombinant AOD was subsequently purified through affinity chromatography and enterokinase cleavage, resulting in the final yield of 216 mg/L and a purity exceeding 93%. The minimum inhibitory concentrations (MICs) of AOD against Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus galactis ranged from 4 to 8 μg/mL. Moreover, time-killing curves indicated that AOD achieved a bactericidal rate of 99.9% against the clinical strain S. epidermidis G-81 within 0.5 h at concentrations of 2× and 4× MIC. Additionally, the activity of AOD was unchanged after treatment with artificial gastric fluid and intestinal fluid for 4 h. Biocompatibility testing demonstrated that AOD, at a concentration of 128 μg/mL, exhibited a hemolysis rate of less than 0.5% and a cell survival rate of over 83%. Furthermore, AOD’s in vivo therapeutic efficacy against mouse subcutaneous abscess revealed its capability to restrain bacterial proliferation and reduce bacterial load, surpassing that of antibiotic lincomycin. These findings indicate AOD’s potential for clinical usage.

• Publisher: MDPI AG
• Subject: abscess / Affinity chromatography / American Oyster Defensin (AOD) / Amino acids / Antibacterial agents / antibacterial properties / Antibiotics / antimicrobial mechanism / Antimicrobial peptides / Bacteria / Bacterial infections / Biocompatibility / Cell division / Cell survival / cell viability / Chemical properties / Chromatography / Crassostrea / Crassostrea virginica / Drug therapy / E coli / enteropeptidase / fermenters / Fusion protein / gastric juice / Gram-positive bacteria / Haemolysis / hemolysis / Hydrophobicity / In vivo methods and tests / intestinal secretions / Lincomycin / Marine molluscs / mice / microbial load / Molecular weight / Oysters / Peptides / Pharmaceutical research / pharmacodynamics / Production processes / Proliferation / protein content / Proteins / Recombinants / Shellfish / small fusion tag / Staphylococcal infections / Staphylococcus aureus / Staphylococcus epidermidis / Staphylococcus infections / Streptococcus / Survival / survival rate / therapeutics / Yields