Heterologous expression of the novel dimeric antimicrobial peptide LIG in Pichia pastoris

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Antimicrobial peptides (AMPs) are crucial for innate immunity.
• The fusion peptide LI combines human LL37 and bovine Indolicidin, retaining antimicrobial properties and cell selectivity.
• Developing efficient expression systems for AMPs is essential for their therapeutic applications.

Purpose of the Study:

• To construct and express a dimeric form of the antimicrobial peptide LI (LIG) in Pichia pastoris.
• To evaluate the antimicrobial activity, hemolytic activity, and stability of the recombinant dimeric peptide (rLIG).
• To explore a novel strategy for producing dimeric AMPs using the P. pastoris expression system.

Main Methods:

• Ligation of LI peptides using a Gly-Ser-Gly (G-S-G) linker to form dimeric LIG.
• Construction of the Pichia pastoris (P. pastoris) expression vector pPIC9K-6×His-3×FLAG-LIG.
• Optimization of recombinant protein expression in P. pastoris GS115, including induction time, methanol concentration, and initial pH.
• Purification of recombinant LIG (rLIG) and assessment of its antimicrobial and hemolytic activities, as well as salt ion stability.

Main Results:

• The P. pastoris GS115 system achieved a peak total protein expression of 189.6 mg/L for LIG.
• 5.9 mg/L of purified recombinant LIG (rLIG) was obtained after enterokinase digestion.
• rLIG exhibited high antimicrobial activity and low hemolytic activity.
• Dimeric LIG showed comparable antimicrobial activity and enhanced salt ion stability compared to monomer LI.

Conclusions:

• The successful expression of dimeric antimicrobial peptide LIG in P. pastoris provides a viable method for AMP production.
• Dimeric LIG demonstrates potent antimicrobial efficacy and stability, suggesting potential for therapeutic applications.
• This study offers valuable insights into optimizing AMP expression in P. pastoris systems.