Systems are convenient single-tube, coupled transcription/translation reactions for eukaryotic cell-free protein expression. This in vitro approach provides quick access to soluble, functional proteins of interest that are post-translationally modified without an additional protein purification step. The cell-free expression system is supplemented with a prokaryotic phage RNA polymerase combined with eukaryotic extracts and primed with exogenous DNA or PCR-generated DNA containing the T7, SP6 or T3 promoter for in vitro protein synthesis (Figure 1). Tn
Systems have great flexibility in design, with an open environment for protein synthesis and a high yield expression output—demonstrating the potential for many downstream applications.
Figure 1. Cell-free expression using the TnT® Systems.
Downstream Applications and Beyond
These systems were first used to determine if a gene or open reading frame (ORF) was present (1–3). The method uses a TnT® System to express a translated protein sequence from DNA or RNA. The protein products are analyzed using methods such as immunoprecipitation, western blot analysis or SDS-PAGE. Resulting proteins are then characterized by size and structure and correlated to the size and sequence of a given gene or ORF.
Additional applications were developed, including use in protein:protein interaction studies. One common method to detect these interactions is the glutathione-S-transferase (GST) pull-down assay (4–6). In this method, one protein partner is expressed and purified from E. coli as a GST fusion protein. The other protein partner is expressed in a TnT® System and labeled with [35S]methionine to be used as a probe for detection of the interaction. The translated products are combined in a mixture, then analyzed by SDS-PAGE and autoradiography to identify the bound protein:protein complex.
In addition to protein:protein interactions, the TnT® System can be utilized to study protein:DNA and protein:RNA interactions. For example, DNA-binding proteins—such as transcription factors—commonly bind to single- or double-stranded DNA. These proteins can be analyzed for their ability to bind to specific sequences on radiolabeled oligonucleotides. Bound and non-bound products are compared using an electrophoretic mobility shift assay, or EMSA (7–9). Nucleic acids bound to protein have less mobility through a gel matrix when compared to non-bound DNA or RNA.
Given the flexibility of the TnT® System, this approach has expanded beyond the methods described above. Here we describe several novel applications across a variety of research areas.
Detection of Autoantibodies in Serum
Antibodies specific for the H+, K+-ATPase proton pump of gastric parietal cells are diagnostic markers in patients with autoimmune gastritis. However, there have been few studies regarding the diagnostic accuracy of serological screening. A recent study used a luminescent immunoprecipitation system (LIPS) to detect autoantibodies for the ATP4A and ATP4B subunits of the proton pump, thought to be the primary antigens in autoimmune gastritis (10).
To detect autoantibodies against the protein subunits, the coding sequences for the ATP4A and ATP4B genes were cloned into a modified pCMVTnT® Vector (Cat.# L5620) containing a luciferase reporter. Recombinant luciferase antigens were expressed in vitro using the TnT® SP6 Quick Coupled Transcription/Translation System (Cat.# L2081). Aliquots of expressed protein were added to human serum samples from patients with and without gastritis.
To recover and detect immune complexes, Protein-A-sepharose was added to bind to the antibodies, followed by a luciferase assay using Renilla or Nano-Glo® Luciferase Assay substrates (Cat.# E2810 and Cat.# N1110). When compared to commercial immunoassays, the LIPS assay resulted in higher sensitivity and comparable specificity, demonstrating its value as a diagnostic screening tool.
Characterization of deSUMOylation Activity
SUMOylation is a post-translational modification process that affects protein function. For example, the PML protein is SUMOylated to form structures called PML bodies which aid in antiviral functions during infection.
Human cytomegalovirus (HCMV) causes viral infections characterized by latent reactivation of the virus throughout the host’s lifetime. A recent study demonstrated that the viral gene product LUNA is responsible for deSUMOylation of the PML protein, which allows latent reactivation of HCMV (11).
One of several assays to characterize the deSUMOylase activity of the LUNA protein utilized cell-free expression with a TnT® T3 Coupled Reticulocyte Lysate System (Cat.# L4950). PML was subjected to SUMOylation reactions in vitro. The SUMO-modified PML was then incubated with various LUNA constructs and a buffer-only control and the samples were analyzed by western blot. The presence of LUNA resulted in a decrease of total PML, and specifically a loss of SUMOylated PML protein. These data help further the understanding of how latent viral infections like HCMV can persist and contribute to disease outcomes.
Assessment of Long Non-Coding RNA-Mediated Translation Inhibition
Long non-coding RNAs are defined by a length greater than 200 nucleotides, and they are not translated into protein. They play an active role in regulating gene expression during transcriptional events, however, these transcripts have also been found to inhibit translation by binding to translational machinery proteins. A recent study observed how this RNA-mediated translation inhibition is controlled through regulatory proteins (12).
BC200 RNA inhibits translation in nerve cells, as well as certain cancer cells and tumors. To characterize how this inhibition could be controlled, a variety of RNAs were transcribed/translated using the TnT® T7 Coupled Reticulocyte Lysate System (Cat.# L4610). The reaction mixes included BC200 RNA and GST fusion proteins for hnRNP-E1 and hnRNP-E2; the constructs utilized luciferase proteins to monitor expression levels. hnRNP-E1 and hnRNP-E2 interacted with BC200 RNA and expression was observed, demonstrating that the proteins can regulate BC200 RNA-mediated inhibition activity.
Identification of Antisense Oligonucleotides to Inhibit Translation of Viral Proteins
Ebola virus is a potentially life-threatening pathogen, depending on the strain and severity of the outbreak. Currently, there is no approved vaccine to treat the disease, though there are several promising candidates in clinical trials. Locked nucleic acid-modified antisense oligonucleotides (LNA ASOs) are an attractive therapeutic strategy due to their stable properties and specific binding affinity toward target protein sequences.
In a recent study, LNA ASOs were developed to inhibit translation of important viral proteins to prevent viral replication and infection (13). Luciferase gene fusion reporters for several Ebola virus proteins were incubated with a variety of LNA ASOs using the TnT® T7 Quick Coupled Transcription/Translation System (Cat.# L1170). The effects were characterized by analyzing luciferase activity.
The LNA ASOs effectively targeted two Ebola virus proteins, NP and VP24, as well as the human intracellular host protein Niemann-Pick C1, which is exploited for viral entry into infected cells. Protein translation was inhibited by LNA ASO binding, suggesting a potential therapeutic strategy to prevent viral infection.
In Vitro Ubiquitination Assay
p53 is a tumor suppressor gene encoding for a protein that regulates the cell cycle. In vivo, p53 is continually ubiquitinated by the protein MDM2, a process that ends in protein degradation. The regulation of p53 is important, because replication of damaged DNA can result in uncontrolled cell growth and cancer.
PIG3 is another protein that contributes to cell cycle regulation by producing reactive oxygen species (ROS) that mediate p53-induced apoptosis. It is also involved in checkpoint signaling and DNA damage response. Recently, PIG3 was found to regulate p53 stability by suppressing MDM2-mediated ubiquitination (14).
TnT® Systems were used to translate p53, MDM2 and PIG3 proteins using appropriate expression vectors. These proteins were then used for an in vitro ubiquitination assay to characterize the effect of PIG3 on p53 ubiquitination by MDM2. The recombinant p53 and MDM2 proteins were mixed with ubiquitin and activating enzymes. In the absence of PIG3, p53 was ubiquitinated by MDM2. When PIG3 was added, ubiquitination was suppressed, indicating that PIG3 interacts directly with MDM2 to suppress p53 ubiquitination.
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- You, J. et al. (2006) Kaposi’s sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with bromodomain protein Brd4 on host mitotic chromosomes. J Virol. 80, 8009–19.
- Zhang, D. et al. (2011). Arginine and Glutamate-rich 1 (ARGLU1) interacts with mediator subunit 1 (MED1) and is required for estrogen receptor-mediated gene transcription and breast cancer cell growth. J. Biol. Chem. 286, 17746–54.
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- Saeki, M. et al. (2011) Functional analysis of genetic variations in the 5’-flanking region of the human MDR1 gene. Mol. Genet. Metab. 102, 91–8.
- Lahner, E. et al. (2017) Luminescent Immunoprecipitation System (LIPS) for detection of autoantibodies against ATP4A and ATP4B subunits of gastric proton pump H+, K+-ATPase in atrophic body gastritis patients. Clin. Transl. Gastroenterol. 8, e215.
- Poole, E. et al. (2018) A virally encoded DeSUMOylase activity is required for cytomegalovirus reactivation from latency. Cell Reports. 24, 594–606.
- Jang, S. et al. (2016) Regulation of BC200 RNA‐mediated translation inhibition by hnRNP E1 and E2. FEBS Letters. 591, 393–405.
- Chery, J. et al. (2018) Development of locked nucleic acid antisense oligonucleotides targeting Ebola viral proteins and host factor Niemann-Pick C1. Nucleic Acid Ther. 28, 273–84.
- Jin, M. et al. (2017) PIG3 regulates p53 stability by suppressing its MDM2-mediated ubiquitination. Biomol Ther. 25, 396–403.