Boosting Expression of a Specifically Targeted Antimicrobial Peptide K in Pichia pastoris by Employing a 2A Self-Cleaving Peptide-Based Expression System

Abstract

Background/Objectives: The current epidemic of drug-resistance bacterial strains is one of the most urgent threats to human health. Antimicrobial peptides (AMPs) are known for their good activity against multidrug resistance bacteria. Specifically targeted AMPs (STAMPs) are a fraction of AMPs that target specific bacteria and maintain the balance of the healthy microbiota of a host. We reported a STAMP Peptide K (former name: peptide 13) for E. coli. The aim of this study was to effectively produce peptide K using methylotrophic yeast Pichia pastoris. Methods: Three inserts (sequence of peptide K (K), two copies of peptide K fused with 2A sequence (KTK), and two copies of peptide K fused with 2A and an extra α mating factor (KTAK)) were designed to investigate the effect of the number of repeats and the trafficking of peptide on the yield. Results: The yield from KTK was the highest-more than two-fold higher compared with K-implying the role of the 2A sequence in heterologous peptide expression apart from the co-translation. Then, the fermentation condition for KTK was optimized. The optimized yield of KTK was 6.67 mg/mL, suggesting the efficiency of the expression system. Selectivity, antibacterial activity, biocompatibility, and the stability of the fermentation product were equivalent to the chemically synthesized peptide. The actional mechanism of the fermentation product included membrane permeabilization and ROS induction. Conclusions: Together, our work provided a new perspective to augment the yield of the antimicrobial peptide in the microbial system, building a technological foundation for their large-scale production and expanding the market application of AMPs.

Keywords: 2A self-cleaving peptide; Pichia pastoris; fermentation condition optimization; selective targeted antimicrobial peptide.

Figure 1

(a) Structure of pPIC9K plasmid and schematic diagram showing the alignment of K, KTK, and KTAK. (b) Agar gel electrophoresis for the recombinant plasmid. M: marker; E: pPIC9K; K: peptide K; T: KTK; A: KTAK. (c) Screening of positive transformants on MD plate. (d) PCR agar gel electrophoresis for monoclonal recombinant GS115 with universal primer AOX.

Figure 2

(a) Time growth of GS115. The culture parameters for the growth curve were a single colony of GS115 with empty vector in BMGY, growing at 30 °C in a shaking incubator 250 rpm. (b) Wet weight of recombinant GS115. (c) Tricine-SDS-PAGE for fermentation products from recombinant GS115. MW: molecular weight; E: supernatant from GS115 containing empty vector; K: supernatant from GS115 containing K; T: supernatant from GS115 containing KTAK; A: supernatant from GS115 containing KTAK. (d) The antibacterial activity of fermentation supernatant. * p < 0.05, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Expression of peptide K at different fermentation time (a) with different methanol concentration (b) and with different pH value (c). M: marker.

Figure 4

(a) Purification of recombinant peptide K. M: marker; K_S: fermentation supernatant; K_P: purified recombinant peptide K. (b) Hemolysis of purified peptide K from KTK. (c) Membrane permeability of recombinant peptide K using NPN method. (d) ROS production induced by recombinant peptide K using DCFH method. ME: melittin.