A Step-by-Step Guide for Biosynthesis of Recombinant Fusion Antimicrobial Peptide and Release of the Active Peptide from Its Fusion Partner by Formic Acid Cleavage

Sivakumar Jeyarajan ^1,2, Sukumar Ranjith ¹, Bency Thankappan ^1,3

⁰Microbial Biotechnology Laboratory, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India.

Methods in Molecular Biology (Clifton, N.J.) +

|

June 18, 2025

View abstract on PubMed

Summary

This study details a cost-effective method for producing short peptides using recombinant fusion in Escherichia coli. Formic acid cleavage simplifies purification, yielding 6.5 mg/L of active peptide.

Area Of Science

Biotechnology
Molecular Biology
Protein Chemistry

Background

Recombinant protein production is crucial for peptide therapeutics.
Traditional peptide purification methods can be complex and costly.
Fusion partners are often used to facilitate peptide expression and purification.

Purpose Of The Study

To present a simplified protocol for cloning and expressing short peptides as fusion proteins.
To demonstrate the efficacy of formic acid cleavage for releasing active peptides.
To optimize the purification and characterization of recombinant peptides.

Main Methods

Gene cloning of short peptides with a fusion partner.
Biosynthesis of recombinant fusion peptide in Escherichia coli.
Peptide separation via formic acid cleavage, purification, and characterization.

Main Results

A cost-effective method for releasing active peptides from fusion partners was established.
The procedure simplifies peptide purification by avoiding denaturant removal steps.
Achieved a yield of 6.5 mg/L of recombinant fusion peptide.

Conclusions

Formic acid cleavage offers an advantageous alternative to enzymatic cleavage for peptide release.
This protocol streamlines recombinant peptide production, reducing time and labor.
The method is effective for producing purified short peptides, though challenges in acid digestion are noted.

Keywords:

Cloning Formic acid cleavage Genetic engineering Protein expression optimization SDS-PAGE Therapeutic antimicrobial peptides Thioredoxin