Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization.

Baseer Ahmad,Zhang Lulu,Zhang Rijun,Si Dayong,Quratulain Hanif,Wei Xubiao,Muhammad Shahid

doi:10.3389/fimmu.2019.01365

Abstract

This study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37Tα1 in PpICZαA vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37Tα1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37Tα1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-α, IL-6, IL-1β, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P. pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37Tα1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity.

Highlights

Antimicrobial peptides (AMPs) are important components of the innate immune response against microbes
Hybrid peptide gene was amplified through PCR and 129 bp DNA fragment encoding a C-terminal 6× His tag which was attached with Kpn I and Xba I restriction enzyme site at it’s 5′ and 3′ end, respectively, was inserted into pUC57 vector
This fragment was double-digested with restriction enzymes (Kpn I and Xba I) and was a frame clone to attach to the 3′ end of the α-factor secretion signal, downstream alcohol oxidase 1 (AOX1) promoter of the expression plasmid pPICZαA to result in a recombinant vector named pPICZαA-LL-37Tα1

Summary

Introduction

Antimicrobial peptides (AMPs) are important components of the innate immune response against microbes. AMPs that bind to LPS and neutralize its effect could have potential clinical applications [6, 7]. Antibiotics can increase the release of bacterial LPS by killing bacteria and activate the immune system to secrete cytokines and produce lethal shock [11]. LPS is an integral component of Gram-negative bacteria’s outer membrane, that is released after lysis of bacteria and leads to a number of pathophysiological conditions like fever, leukopenia, intravascular coagulation, and sepsis [12, 13]. Over the past decades, there has been a dire need for new antiinflammatory peptides that have both antibacterial and LPS neutralizing activities [10]

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Conclusion