Sortase A Fusion Expression and mIFc2 Co-Expression of Bovine Lactoferricin and Analysis of Its Antibacterial Activity

Author:

Hsu Chao-YuORCID,Hsieh Chung-Yiu,Yang Cheng-YaoORCID,Chang Yu-Kang,Shih Wen-Ling,Yeh Chuan-Ming,Hu Nien-JenORCID,Chen Ming-Shan,Nielsen Brent L.ORCID,Liu Hung-JenORCID

Abstract

The coding region for the sortase A (SrtA) of Staphylococcus aureus was fused at the N-terminus of LfcinB. The SrtA-LfcinB fusion protein in E. coli C43(DE3) was expressed with the expected sizes of 21 kDa and 38 kDa by pET21b-SrtA-LfcinB and pET32-1SrtA-LfcinB constructs, respectively. Increased levels of the TrxA-His-SrtA-SrtA-LfcinB fusion protein were detected by the pET32-3SrtA-LfcinB construct having three expression cassettes. LfcinB is released from the expressed SrtA-LfcinB protein by SrtA self-cleavage which is induced in the presence of Ca2+. The antibacterial activity was detected after SrtA-mediated cleavage of LfcinB. Furthermore, to reduce the antimicrobial peptide toxicity to the E. coli host, the human interferon-γ (hIFN-γ) sequences were mutated into a negatively charged mIFc2 protein (7 kDa), which was co-expressed with LfcinB in an insoluble form. The yield of LfcinB was elevated while changing the gene order of LfcinB and mIFc2 (pET21b-fLfcinB-bmIFc2). Furthermore, increased levels of LfcinB were detected using the pET21b-(fLfcinB-bmIFc2)2 construct. To increase the dissolution rate of inclusion bodies, inclusion bodies treated with different temperatures and pH and resuspended in different volumes of 50 mM Tris-HCl were assayed. Our results reveal that heat-treated LfcinB/mIFc2 inclusion bodies at 90 °C, pH 10, and 16X resuspended volumes have the best resolubilization rate. This work suggests that the mIFc2 co-expression system shows higher efficiency for LfcinB production than the SrtA fusion system. The expressed LfcinB from the mIFc2 co-expression system exhibits excellent broad-spectrum antibacterial activities against thirteen Gram-negative and ten Gram-positive bacteria species with a range of minimum inhibitory concentrations (MIC) between 37–150 ug/mL.

Funder

Tungs’ Taichung MetroHarbor Hospital

Taichung Veterans General Hospital

iEGG and Animal Biotechnology Center

Ministry of Science and Technology in Taiwan

Publisher

MDPI AG

Subject

Process Chemistry and Technology,Chemical Engineering (miscellaneous),Bioengineering

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