Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System

Abstract

In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells.

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

“…To the best of our knowledge, this is the first reported synthesis of active HBCM2 from a recombinant source. Our reported yield is within the range of those observed for the calmodulin fusion protein system (0.1–4.6 mg/L depending on the peptide), which was recently identified as a rationally designed carrier protein for AMPs (Ishida et al, ). In this system, the two domains of calmodulin were shown to effectively wrap around the fused AMP through charge interactions to occlude the AMP and thus reduce its toxicity and prevent proteolysis.…”

“…These carrier proteins aid protein purification and are later cleaved from the AMPs using chemical methods or specific proteases. However, selection of the optimal carrier protein for a given AMP is still empirical (Parachin et al, ) and carrier proteins that are rationally designed to reduce toxicity and proteolytic susceptibility while maintaining solubility are limited (Ishida, Nguyen, Gopal, Aizawa, & Vogel, ).…”

The Calcium-Dependent Switch Helix of L-Plastin Regulates Actin Bundling

“…SPR experiments were carried out to investigate the interactions between the EF domain of LPL (EF construct) and various synthetic CaM-binding peptides. Since the EF structure of LPL is very similar to one of the two CaM domains, we decided to study two typical CaM-binding peptides, CaMKIp and smMLCKp, as well as the cytotoxic peptide melittin, which is also known to bind to CaM 56 57 ( Supplementary Fig. S7 ).…”

A Dynamic Overview of Antimicrobial Peptides and Their Complexes

“…To apply advanced NMR techniques, relatively large amounts of isotope-labeled proteins containing 13 C, 15 N, or 2 H are desired [ 30 , 31 ]. Therefore, a number of methods for producing recombinant AMPs in bacteria have been developed [ 32 , 33 , 34 ]. Recombinant AMPs can be challenging to express because they can be toxic to their host bacteria and susceptible to proteases.…”