Concept study to develop a platform process for the production of antimicrobial peptides/proteins

Abstract

The increasing prevalence of antibiotic-resistant pathogens has created a demand for alternative treatments. However, the production of antimicrobial peptides and proteins (AMPs) in sufficient quantities for therapeutic use remains a significant bottleneck. This study introduces an innovative platform technology for the heterologous production, purification, and detection of AMPs. The technology is based on a small fusion tag, called the C-tag, composed of four amino acids (EPEA) fused to the C terminus of AMPs. Notably, using a complex model AMP, we demonstrated for the first time that the C-tag enhances the expression of this AMP in Escherichia coli compared to the unmodified peptide. Further process intensification increased the yield by nearly 30-fold compared to previous attempts with the same AMP. The primary advantage of the tag is apparent during product purification, leading to > 85% recovery and ~80% purity in a single step without compromising product activity, thus eliminating the need to remove the tag in a cost-intensive additional process step. In the subsequent parts of this study, we introduce, for the first time, an analytical immunoaffinity chromatography based on the C-tag. A detailed exploration of the critical process parameters using statistical optimization techniques enabled us to achieve 98.8 ± 0.1% analyte recovery. Validation of the method indicated high specificity, linearity, accuracy, and precision, underscored by a short sample analysis time, enabling high-throughput sample screening. Further characterization of the method revealed its limited ability to detect AMPs in acidic samples (pH < 2). Pretreatment strategies to overcome these limitations are discussed. Consequently, this study introduces a quantitative C-tag platform for the in-process monitoring of AMPs.