Cell-free production of a functional oligomeric form of a Chlamydia major outer-membrane protein (MOMP) for vaccine development

Abstract

Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. infection, therefore, constitutes a significant public health threat, underscoring the need for a -specific vaccine. strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an -based cell-free system to express a MOMP protein from the mouse-specific species (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

“…Using E. coli-based CFPS, mMOMP was synthesized in a native trimeric form in the presence of nanolipoproteins (NLPs) with a yield of around 1.5 mg/mL. When injected into mice in the presence of an adjuvant, the protein elicited an enhanced humoral immune response [126]. This method of synthesizing and simultaneous incorporation of antigens into NLPs using a CF approach is a promising method for future vaccine development.…”

Telodendrimers: Promising Architectural Polymers for Drug Delivery

“…Its use is limited by poor stability. PEG-cholic acid-based telodendrimer–nanolipoprotein particles were used in this study, and the cell-free system expressed mMOMP multimers, similar to the native MOMP protein, demonstrating that this approach using telodendrimers can be used to support antigens, which is otherwise difficult [ 205 ].…”

Cell-free expression of the outer membrane protein OprF of Pseudomonas aeruginosa for vaccine purposes

“…In the presence of liposomes or nanodiscs, cell-free systems allow the membrane protein to be translated and spontaneously inserted into the liposomal membrane in a one-step reaction, skipping the laborious solubilization, purification, and proteoliposome reconstitution steps required when using cellular expression systems (Liguori et al, 2016(Liguori et al, , 2015Liguori and Lenormand, 2009;Sachse et al, 2014). Finally, CFPS systems also enable rapid screening of different liposomal compositions, therefore increasing the likelihood of reconstituting the membrane entire protein in its active form (Kimura-Soyema et al, 2014) and presenting native antigens (He et al, 2017).…”