Antimicrobial peptides (AMPs) are strong therapeutic candidates against antibiotic -resistant, pathogenic bacteria, but they are difficult to produce in cell-based systems in sufficient quantities because they are cytotoxic and susceptible to proteolytic degradation. They are often chemically synthesized using liquid and/or solid phase peptide synthesis, which can be very expensive particularly for longer length peptides and suffer from additional disadvantage of limited yields. Alternatives such as cell-free protein synthesis systems (cell free expression, www.lifetechnologies.com) are also expensive due to expensive raw materials and usually provide lower yields compared to in vivo (e.g., E. coli) production systems.
Researchers at LLNL have developed a novel method to express and purify significant quantities of AMPs. AMP is fused to the N-terminus of a self-assembling protein called encapsulin from Thermotoga maritima, which forms protein cages with 60 monomer units. N-terminal fusion of the peptide to encapsulin results in encapsulation of the peptide within the protein cage, which prevents cytotoxicity of the peptide to the host and protects the peptide from host proteolysis, ultimately enabling high cellular production levels. In order to isolate the peptide from the protein cage, specific protease cleavage sites (e.g., TEV protease sites) are engineered into the encapsulin cage. Upon treatment with the appropriate protease, the peptide is released and can be isolated for a designated purpose.
For additional technical information refer to the article “Re-directing bacterial microcompartment systems to enhance recombinant expression of lysis protein E from bacteriophage ϕX174 in Escherichia coli”.
LLNL's proprietary method enables expression of peptides within encapsulin protein cages in an E. coli host-based expression system. The cages prevent toxicity of the peptide to the host organism as well as protect the peptide against non-specific proteases, thus enabling high peptide production levels. This system also has high fidelity compared to chemical synthesis and therefore may reduce downstream purification costs.
- Production and isolation of toxic peptides from a cell-based expression system;
- Production and isolation of protease-sensitive peptides from a cell-based expression system;
- Drug delivery of encapsulated peptides for therapeutic applications
LLNL has filed for patent protection for this technology:
U.S. Patent Application 15/178454, “Engineering Bacterial Systems to Shield Toxicity During Non-Native Protein Expression and Purification” (LLNL internal case #IL-13008)
U.S. Provisional Application 62/598984, “Engineered microcompartment protein and related methods and systems of engineering bacterial systems for non-native protein expression and purification” (LLNL internal case #IL-13262).