Date Published: April 25, 2019
Publisher: Public Library of Science
Author(s): Srujan Kumar Dondapati, Georg Pietruschka, Lena Thoring, Doreen A. Wüstenhagen, Stefan Kubick, Salvatore V Pizzo.
The Toll-like receptor family belongs to the group of pathogen recognition receptors which is responsible for the discrimination of self and non-self pathogen-associated molecular patterns (PAMP’s). Toll-like receptors play an important role in the innate immunity and defects in protein expression or polymorphism is linked to various diseases such as Systemic Lupus Erythematosus (SLE). The elucidation of the underlying mechanism is crucial for future treatment and therapeutics of toll-like receptor linked diseases. Herein, we report the cell-free synthesis of human Toll-like receptor 9 (hTLR9) using CHO lysate and the continuous exchange cell-free (CECF) synthesis platform. The functionality of this protein was demonstrated by an ELISA binding assay using the ectodomain of TLR9 (TLR9-ECD).
The Toll-like receptor (TLR) family is an important group of pattern recognition receptors (PRRs) through which innate immunity recognizes various pathogen-associated molecular patterns (PAMPs) derived from different invasive microorganisms . Toll-like receptors are type I transmembrane proteins consisting of an ectodomain, one transmembrane domain and the cytosolic Toll-interleukin 1 (IL1) receptor (TIR) domain. TLRs recognize pathogen-derived ligands by their ectodomains . The structural motif of the ectodomain (ECD) contains leucine rich repeats (LRR) responsible for PAMP recognition . Among the TLR family, TLR9 receptors are localized in the intracellular vesicles particularly in the endoplasmic reticulum (ER) in resting cells, and in endosomes upon stimulation by ligands . TLR9 specifically recognizes single stranded DNA containing a CpG motif [5–7]. Recently, the crystal structure revealed the binding mechanism of mouse TLR9 to oligonucleotides (ODN) . Before, the binding mechanism of human TLR9 (hTLR9) was proposed in an in-silico approach . Further investigation of TLR9 is needed since targeting TLR9 receptors and modulating TLR9 signaling have emerged as important strategies as it is involved in autoimmune diseases such as systemic lupus erythematosus (SLE) [10–12]. A number of oligonucleotides modulating TLR9 immune response are in the pipeline and are in the different clinical trial phases for use in SLE treatment. Several synthetic oligonucleotide (ODN) agonists for TLR9 are currently in development for the treatment of cancer . Especially, developing a faster method of TLR9 synthesis and characterization is crucial providing molecular details of the binding mechanism thereby enabling new opportunities for future drug discovery.
Herein, we present for the first time the synthesis of hTLR9 in a cell-free system using CHO lysates. With this synthesis platform it was possible to obtain protein yields of hTLR9 with up to 680 μg/ml in the microsomal fraction (Fig 2A, Table 2). CHO cell lysate was usually used for fundamental understanding of mRNA translation [25,26]. Furthermore, Broedel et al. demonstrated in 2013 for the first time the cell-free synthesis of heparin-binding EGF-like growth factor receptor, epidermal growth factor receptor, and erythropoietin using the CHO system . By fusion of an internal ribosomal entry site (IRES) of the Cricket Paralysis Virus (CrPV), it was possible to synthesize proteins in a cell-free dependent manner using a mammalian cell lysate. It must be emphasized that for optimal high yield protein synthesis it was also necessary to substitute the normally used AUG start codon by a GCU codon . By this substitution, an increase of luciferase synthesis could be achieved in a batch based system using the CHO lysate [20,27]. Further, the synthesis has been performed in a coupled system, enabling simultaneous transcription and translation, to save time and labor compared to the linked system . Using the coupled system has also the advantage that it can be implemented in a variety of other cell-free platforms [23,30,31]. Furthermore, we used the continuous exchange cell-free (CECF) synthesis technology for high yield protein production. The advantages of CECF in CFPS were extensively discussed by Thoring et al [21,24]. Prior to high yield protein synthesis, several optimizations are mandatory. It was shown that the DNA template has an influence on protein yield. Using linear DNA templates resulted in a lower protein yield compared to a circular DNA template . Further optimization could be achieved by adjusting the T7 polymerase concentration in the synthesis reaction as well as the addition of molecular crowding reagents like PEG . Additionally, the optimization of the magnesium concentration has a significant impact in CFPS. By adjustment of the magnesium concentration from 3.9mM used for batch reactions to 22.5 mM, a 3-fold increase in protein yield could be obtained . In this work, we focused mainly on the optimization of the reaction time and reaction temperature. We could show that a prolonged reaction time is linked to an increase in protein yield as demonstrated for the full-length construct NCM-TLR9-His10 (Fig 1C). However, the maximum yield was achieved after 48h and further increase in reaction time did not result in a higher protein yield. Next, the elucidation of the proper reaction temperature was another crucial parameter for high yield CFPS (Fig 1A).