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This chapter explains the step-by-step protocol for cloning the gene for short peptides with a fusion partner and the biosynthesis of recombinant fusion peptide in Escherichia coli followed by the separation of the active peptide by formic acid cleavage and its purification and characterization. The advantage of formic acid cleavage is its cost-effectiveness in releasing the active peptides from the fusion partner compared to enzymatic cleavage and the nonrequirement of folded conformation for acid cleavage. This reduces the time, labor, and steps required for removing the denaturant, thus simplifying the procedure. With this procedure, 6.5 mg/L of recombinant fusion peptide was obtained. The challenges associated with acid digestion are also mentioned in this protocol.

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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

  • 实验方案

  • 发表日期：

    2025年

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  • Sivakumar Jeyarajan,
  • Sukumar Ranjith,
  • Bency Thankappan,
  • Anbarasu Kumarasamy

摘要

This chapter explains the step-by-step protocol for cloning the gene for short peptides with a fusion partner and the biosynthesis of recombinant fusion peptide in Escherichia coli followed by the separation of the active peptide by formic acid cleavage and its purification and characterization. The advantage of formic acid cleavage is its cost-effectiveness in releasing the active peptides from the fusion partner compared to enzymatic cleavage and the nonrequirement of folded conformation for acid cleavage. This reduces the time, labor, and steps required for removing the denaturant, thus simplifying the procedure. With this procedure, 6.5 mg/L of recombinant fusion peptide was obtained. The challenges associated with acid digestion are also mentioned in this protocol.