Recombinant expression, purification and PEGylation of Paneth cell peptide (cryptdin-2) with value added attributes against Staphylococcus aureus

Navneet Kaur,Amrita Kaur,Rahul Dilawari,Praveen Rishi,Girish Sahni

doi:10.1038/s41598-020-69039-2

Navneet Kaur, Amrita Kaur + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-69039-2

Copy DOI

Abstract

Cryptdins are disulfide-rich cationic antimicrobial peptides secreted by mouse Paneth cells and are known to exhibit potent antimicrobial activity against various deadly pathogens. Keeping in view the extremely low yield obtained from mouse Paneth cells and high cost of synthetic peptide(s), herein, we have attempted to produce cryptdin-2 in Escherichia coli using recombinant technology. To avoid lethal effects of peptide on the host cells, cryptdin-2 was expressed as a fusion protein with thioredoxin as fusion partner which yielded 40 mg/L protein in the soluble fraction. Subsequently, mature cryptdin-2 was cleaved from the fusion partner and purified by cation exchange chromatography. Since conjugation of poly(ethylene) glycol (PEG) has been known to improve the biological properties of biomolecules, therefore, we further attempted to prepare PEG-conjugated variant of cryptdin-2 using thiol specific PEGylation. Though the antimicrobial activity of PEGylated cryptdin-2 was compromised to some extent, but it was found to have enhanced serum stability for longer duration as compared to its un-modified forms. Also, it was found to exhibit reduced toxicity to the host cells. Further, its synergism with gentamicin suggests that PEGylated cryptdin-2 can be used with conventional antibiotics, thereby indicating its possibility to be used as an adjunct therapy.

Highlights

IntroductionNative (i.e. isolated from mouse Paneth cells) as well as chemically synthesized cryptdin-2 has been previously reported to possess a relatively broad spectrum antimicrobial activity against various deadly pathogens including S. aureus, Y. enterocolitica and S
Development of antimicrobial peptides (AMPs) as therapeutic agents has long been recognized as an important landmark to address the world wide challenge of microbial resistance towards conventional antibiotics
The recombinant plasmid harboring cryptdin-2 gene fused with N-terminal tag was transformed into E. coli and cryptdin-2 fusion protein was found to be non-toxic to the host bacterium E. coli

Summary

Introduction

Native (i.e. isolated from mouse Paneth cells) as well as chemically synthesized cryptdin-2 has been previously reported to possess a relatively broad spectrum antimicrobial activity against various deadly pathogens including S. aureus, Y. enterocolitica and S. Staphylococcus aureus is an opportunistic pathogen causing a variety of clinical manifestations including nosocomial and community acquired infections ranging from mild skin infections to life-threatening diseases such as meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteraemia and sepsis To combat such lethal infections the scientific community is exploring the possibility of using combination therapy. We carried out PEGylation of the peptide after cysteine substitution mutagenesis to evaluate the effects of PEG-conjugation on the biological attributes of cryptdin-2 and its potential to be used in adjunction with the conventional antibiotics

Methods

Results

Discussion

Conclusion