protein synthesis

Cell Free Application: Characterization of Long Non-coding RNA Inhibition of Transcription

Posted on by Gary Kobs

Long noncoding RNAs have been shown to regulate chromatin states, transcriptional activity and post transcriptional activity (1). Only a few studies have observed long non-coding RNAs modulating the translational process (2). The noncoding RNA BC200 has been shown to inhibit translation by interacting with the translation initiation factors, eIF4A and eIF4B.

To characterize how BC200 translational inhibition could be controlled,  a variety of RNAs were transcribed/translated in vitro using the TNT system (Cat. #L4610) from Promega. To each transcription/translation reaction, BC900 RNA, hnRNPE1 and hnRNE2 proteins were added. Inhibition of BC200 activity was noted when proteins were successful expressed (3).

Literature Cited

  1.  Sosinska, P et.al. (2015) Intraperitoneal invasiveness of ovarian cancer from the cellular and molecular perspective. Ginekol. Pol. 86, 782–86.
  2. Geisler, S. and Coller, J. (2013) RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts. Nat.Rev. Mol. Cell. Bio. 14,699–12.
  3. Jang, S. et. al. (2017) Regulation of BC200 RNA-mediated translation inhibition by hnRNP E1 and E2. FEBS Letters. 591, 393–5.

Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit

Posted on by Gary Kobs
NanoLuc dual reporters
Illustration showing NanoLuc and firefly luciferase reporters.

The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).

In a recent publication (2), this assay was used to assess the presence of autoantibodies against ATP4A and ATP4B subunits of parietal cells H+, K+-ATPase in patients with atrophic body gastritis and in controls. Continue reading “Luciferase Immunoprecipitation System Assay (LIPS): Expression of Luciferase Antigen using TNT Transcription/Translation Kit”

Characterization of Ubiquitination Using Cell-Free Expression

Posted on by Gary Kobs

Ubiquitination refers to the post translational modification of a protein by attachment of one or more ubiquitin monomers. The most prominent function of ubiqutin is labeling proteins for proteasome degradation. In addition to this function ubiquitination also controls the stability, function and intracellular localization of a wide variety of proteins.

Cell free expression can be used to characterize ubiquitation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with E1 ubiquitin activating enzyme, E2 ubiquitin –conjugating enzyme, and ubiquitin). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels.

The following references illustrate the use of cell free expression for this application.

Jung, Y.S. et al. (2011) The p73 Tumor Suppressor Is Targeted by Pirh2 RING Finger E3 Ubiquitin Ligase for the Proteasome-dependent Degradation. J. Biol. Chem. 286, 35388–95.

Su, C-H, et al. (2010) 14-3-3sigma exerts tumor-suppressor activity mediated by regulation of COP1 stability. Cancer. Res. 71, 884–94.

Naoe, H. et al. (2010). The anaphase-promoting complex/cyclosome activator Cdh1 modulates Rho GTPase by targeting p190 RhoGAP for degradation. Mol. Cell. Biol. 30, 3994-05.

de Thonel, A. et al. (2010) HSP27 controls GATA-1 protein level during erythroid cell differentiation. Blood 116, 85–96.

Kaneko, M. et al. (2010) Loss of HRD1-mediated protein degradation causes amyloid precursor protein accumulation and amyloid-beta generation. J. Neurosci. 30, 3924–32.

Cell-Free Expression: Non-Radioactive Detection/Applications

Posted on by Gary Kobs

The Transcend™ Non-Radioactive Translation Detection Systems allow nonradioactive detection of proteins synthesized using cell free expression. Using these systems, biotinylated lysine residues are incorporated into nascent proteins during translation, This biotinylated lysine is added to the translation reaction as a precharged ε-labeled biotinylated lysine-tRNA complex rather than a free amino acid. After SDS-PAGE and electroblotting, the biotinylated proteins can be visualized by binding either Streptavidin-Alkaline Phosphatase (Streptavidin-AP) or Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), followed either by colorimetric or chemiluminescent detection. Typically, these methods can detect 0.5–5ng of protein within 3–4 hours after gel electrophoresis and can be used for a variety of proteomics related applications. Examples include: Continue reading “Cell-Free Expression: Non-Radioactive Detection/Applications”

Protease K Protection Assay: Cell Free Expression Application

Posted on by Gary Kobs

Microsomal vesicles are used to study cotranslational and initial posttranslational processing of proteins. Processing events such as signal peptide cleavage, membrane insertion, translocation and core glycosylation can be examined by the transcription/translation of the appropriate DNA in the TNT® Lysate Systems when used with microsomal membranes.

The most general assay for translocation makes use of the protection afforded the translocated domain by the lipid bilayer of the microsomal membrane. In this assay protein domains are judged to be translocated if they are observed to be protected from exogenously added protease. To confirm that protection is due to the lipid bilayer addition of 0.1% non-ionic detergent (such as Triton® X-100) solubilizes the membrane and restores susceptibility to the protease.

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Many proteases have proven useful for monitoring translocation in this fashion including Protease K or Trypsin.

The following are examples illustrating this application:

  1. Minn, I. et al. (2009) SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans. Mol. Biol. Cell. 20, 4586–95.
  2. Padhan, K. et al. (2007) Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J.Gen.Virol. 88, 3067–77.
  3. Tews, B.A. et al. (2007) The pestivirus glycoprotein Erns is anchored in plane in the membrane via an amphipathic helix. J.Biol.Chem. 282, 32730–41.
  4. Pidasheva, S. et al. (2005) Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia. Hum. Mol. Gen. 14, 1679–90.
  5. Smith, D. et al. (2002) Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation. J. Immun. 169, 99–107.

Cell-Free Protein Synthesis

Posted on by Promega

Cell-free protein synthesis (aka: in vitro translation) refers to protein production in vitro using lysates that provide the cellular machinery necessary for synthesis. Ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation/elongation/termination factors, GTP, ATP, Mg2+ and K+ are among the requirements for a translation system. These are provided by lysates, which can be from prokaryotic or eukaryotic sources, depending on your requirements.

Cell-free protein synthesis is most commonly used for generating protein for study of things like:

Continue reading “Cell-Free Protein Synthesis”