SHTXTHHly, an extracellular secretion platform for the preparation of bioactive peptides and proteins in Escherichia coli

Abstract

Background In previous work, we developed an E. coli extracellular secretion platform XTHHly based on the hemolysin A secretion system. It can produce bioactive peptides with simple purification procedures. However, the wider application of this platform is limited by poor secretion efficiency. Results In this study, we first discovered a positive correlation between the isoelectric point (pI) value of the target protein and the secretion level of the XTHHly system. Given the extremely high secretion level of S tag, we fused it at the N-terminus and created a novel SHTXTHHly system. The SHTXTHHly system significantly increased the secretion levels of antimicrobial peptides (PEW300, LL37, and Aurein 1.2) with full bioactivities, suggesting its excellent capacity for secretory production of bioactive peptides. Furthermore, RGDS, IL-15, and alcohol dehydrogenase were successfully secreted, and their bioactivities were largely maintained in the fusion proteins, indicating the potential applications of the novel system for the rapid determination of protein bioactivities. Finally, using the SHTXTHHly system, we produced the monomeric Fc, which showed a high affinity for Fcγ Receptor I and mediated the antibody-dependent immunological effects of immune cells, demonstrating its potential applications in immunotherapies. Conclusions The SHTXTHHly system described here facilitates the secretory production of various types of proteins in E. coli. In comparison to previously reported expression systems, our work enlightens an efficient and cost-effective way to evaluate the bioactivities of target proteins or produce them. Graphical abstract

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes

“…Melittin was successfully produced in E. coli for the first time using an innovative production platform called THHly, which relies on the HlyA secretion system. In this process, the MET gene was fused with the signal sequence of HlyA (C-terminal), allowing its secretion to be mediated through the accessory proteins hemolysin B (HlyB) and hemolysin D (HlyD) [ 253 ]. This method was recently used for the recombinant production of melittin and other anti-microbial peptides ( Figure 11 ).…”

“…Subsequent induction with arabinose and IPTG leads to the secretion of the protein of interest (POI) into the supernatant. The figure has been adapted from [ 253 ].…”

Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

“…The C-terminally attached His6-tag is the common design for a nity puri cation of the extracellularly produced proteins [6][7][8][9][10][11]. In a few cases, the His6-tag is incorporated between the target protein and Cterminal signal peptide [12], or fused to the N-terminus of the target protein [13,14], for purifying the protein secreted into the culture. The a nity tags including glutathione S-transferase (GST) as the Cterminal fusion partner, and maltose binding protein as the N-terminal carrier are occasionally used for rapid puri cation of the produced proteins of interest via extracellular production [15,16].…”