Cell Free Expression Application: In vitro degradation assay

A protein chain being produced from a ribosome.

A protein chain being produced from a ribosome.

Researchers and clinicians are fairly certain that all cervical cancers are caused by Human Papillomavirus (HPV) infections, and that HPV16 and HPV18 are responsible for about 70% of all cases. HPV16 and HPV18 have also been shown to cause almost half the vaginal, vulvar, and penile cancers, while about 85% of anal cancers are also caused by HPV16.

E6 is a potent oncogene of HR-HPVs, and its role in progression to malignancy continues to be explored. The E6 oncoprotein of HPV can promote viral DNA replication through several pathways. It forms a complex with human E3-ubiquitin ligase E6-associated protein (E6AP), which can in turn target the p53 tumor-suppressor protein, leading to its ubiquitin-mediated degradation. In particular, E6 from HR-HPVs can block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV-infected cells.

In a recent publication a new procedure generated a stable, unmutated HPV16 E6 protein (1). Continue reading

Protein:DNA Interactions—High-Throughput Analysis

Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.

The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Promega). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.

Literature Cited

  1. Ren, B. et al. (2007) Genome-wide mapping of in vivo protein-DNA binding proteins. Science 316, 1497–502.
  2. Garner, M.M. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions. Nuc. Acids. Res. 9, 3047-60.
  3. Glick,Y et al. (2016) Integrated microfluidic approach for quantitative high throughput measurements of transcription factor binding affinities. Nuc. Acid Res. 44, e51.

Moving Out of the Cell: Advantages of Cell-Free Protein Expression

Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in the traditional E. coli expression systems.

Cell-free protein expression offers significant time savings over cell-based expression methods.

Cell-free protein expression offers significant time savings over cell-based expression methods.

Continue reading

Characterizing Unique Protein: DNA Interactions Using Cell-Free Protein Expression

Molecular model of human telomere DNA

Molecular model of human telomere DNA

The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5′-TTAGGGTTAG-3′ and forms a dimer with the TPP1 protein. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5′-TTAGGG whereas OB2 binds to the downstream TTAG-3′ (1,2). Several recent studies from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected (3). A recent reference reexamined the sequence-specificity of the Human POT1 protein (4).
SELEX (Systematic Evolution of Ligands through Exponential Enrichment) was used  to re-examine the DNA-binding specificity of human POT1 (5). Continue reading

Cell-Free Expression Application: Antibody Screening

TestPermissions2Ricin, derived from caster seeds, inhibits protein synthesis by binding to ribosomes, resulting in cell death. The protein is composed of two polypeptide chains: Ricin Toxin A chain and Ricin Toxin B chain. Ricin inhibits protein synthesis very quickly, and the cell or tissue damage begins within several hours. However, signs of poisoning often are not noted before significant damage has been done, making treatment difficult. Therapeutics that either block the ribosome binding site or compete with the toxin for binding are highly desired. Both antibodies and competitive ligands inhibited binding of the toxin to cell membranes.

A recent publication by Dong et al. (1), described a study to investigate the therapeutic effect of mAb 4C13, a monoclonal antibody against ricin. One of first experiments performed was to determine the general effect the inhibition of protein synthesis induced by ricin using cell-free expression.

In the study, the authors used T3 Coupled Reticulocyte Lysate Systems from Promega. Both ricin and mAb were diluted with saline. Aliquots of ricin (80 ng/ml) were mixed with an equal volume mAbs (1.6μg/ml) or saline alone and incubated at 4 °C for 1.5 h. A total volume of 4μl of sample was added into the reaction system (i.e, T3 Coupled Reticulocyte and plasmid DNA containing the lucifersase gene downstream of T3 RNA phage promoter). After incubation at 30 °C for 1.5 h, the products were cooled at −20 °C for 10 min. A total of 5μl of each reactive product containing synthesized luciferase was mixed with 50μl luciferase assay reagent pre-equilibrated to room temperature, and the fluorescence absorbance was measured immediately with the micro-ELISA Reader.

Positive results obtained from this preliminary experiment, led to more thorough experiments to determine the dosage effect using in vivo models (i.e., cell lines and mice) to characterize the cytotoxicity and binding activity of mAb 4C13. The mAB 4C13 was shown to be a effective in the mouse model.

Dong, N. et al. (2015) Monoclonal antibody , mAb 4C13, an effective detoxicant antibody against ricin poisoning. Vaccine 33, 3836–42

Use of Cell-Free Technology to Evaluate Nuclease (TALEN) Activity on Target DNA

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

ImageSource=RCSB PDB; StructureID=1qpf; DOI=http://dx.doi.org/10.2210/pdb1qpf/pdb;

Transcriptional activator-like effector nucleases (TALENs) have rapidly become a technique of choice for precision genome engineering. TALENs are custom-designed nucleases that consist of a modular DNA-binding domain fused to a monomeric, C-terminal FokI nuclease domain (1). TALENs work in pairs and are designed to recognize and bind to tandem-oriented sequences in genomic DNA, separated by a short spacer (15–30 bp). TALEN binding causes dimerization and activation of the FokI nuclease domains, which results in cleavage of the DNA within the spacer region. Small insertions or deletions (indels) are frequently introduced at this site, as the result of errors made during DNA repair by nonhomologous end-joining (NHEJ). These indels can be up to several hundred base pairs in length and result in frameshift mutations that lead to the production of truncated or nonfunctional proteins.

Successful use of TALENs for inducing targeted mutations has been reported in many conventional models, for example: mice, Xenopus and D. melanogaster. TALENs are also reported to be functional in a variety of other invertebrate arthropods, including mosquitos,silkworm and cricket. A recent publication (2) illustrates the use of TALEN technology for the genetic manipulation in P. dumerilii (marine ragworm). Continue reading

Lost in Translation? Tips for Preparing RNA for in vitro Translation Experiments

A protein chain being produced from a ribosome.

A protein chain being produced from a ribosome.

Synthesizing proteins in vitro through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function.  If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale).  There are many systems out there, and knowing which to use can sometimes be difficult.  Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA.  But when you want to make your own RNA templates to add to lysates, then there are additional concerns.

Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason.  Extracting it requires the utmost care in technique and elimination of nucleases.  Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease).  Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.

The first step for using cell-free expression systems with RNA templates is to make the RNA.  Here are some tips that will ensure success. Continue reading

Cell-free expression application: Screening for successful oligo-mediated knockdown design

800px-ZebrafischAlthough previous references have provided data regarding the potential oncogenic role of the gene ETV7, there has been minimal investigation as to its physiological role.
In the following reference, Quintana, A. et al. (2014) Disease Models & Mechanisms 7, 265–70, zebrafish were used as in vivo model system to characterize ETV7.

One key experiment required the morpholino-oligonucleotide -mediated knockdown of in vivo ETV7. Two independent morpholinos were designed: one that inhibited translation and the other that inhibited proper splicing of exon 3. The efficacy of the translation –blocking morpholino was assessed with cell free expression of ETV7-tagged with hemagglutinin (HA).

Western blot performed with anti-HA antibodies determined the extent of the knockdown compared to a control containing no morpholino added. Once an efficient design was determined via cell-free expression screening, it was used for in vivo experiments. In conjunction additional other techniques, concluded that ETV7 is essential for normal red blood cell development.

Cell-free Expression: A System for Every Need

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Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in traditional E. coli expression systems.

In his webinar, “In vitro, cell-free protein expression–How it helps speed up your research”, Gary Kobs offered an overview of the different cell-free expressions systems offered by Promega and highlighted what needs the different systems best address. He discussed different applications of cell-free expressed proteins and highlighted the combined uses of the HaloTag® Technology with cell-free protein expression. Continue reading

Rabbit Reticulocyte Lysate Translation Systems: Tools for the analysis of translational regulation

TEM of Norovirus particles. Photo Credit: Charles D. Humphrey, Centers for Disease Control and Prevention

TEM of Norovirus particles. Photo Credit: Charles D. Humphrey, Centers for Disease Control and Prevention

Rabbit Reticulocyte Lysate Translation Systems are used in the identification of mRNA species, the characterization of their protein products and the investigation of transcriptional and translational control. Rabbit Reticulocyte Lysate is prepared from New Zealand white rabbits. After the reticulocytes are lysed, the extract is treated with micrococcal nuclease to destroy endogenous mRNA and thus reduce background translation to a minimum.

Untreated Lysate is prepared from New Zealand white rabbits in the same manner as treated lysates with the exception that it is not treated with micrococcal nuclease. Unlike a coupled system that initiates transcription/translation from DNA, the RNA-based rabbit reticulocyte can be used for the direct investigation of transcriptional/translational control and the replication of RNA-based viruses.


References

Characterization of translation regulation (i.e., UTRs, Capping, IRES)

  1. Nguyen, H-L .et al. (2013) Expression of a novel mRNA transcript for human microsomal epoxide hydrolase is regulated by short reading frames within it 5’ –untranslated region. RNA. 19, 752–66.
  2. Wei, J. et al. (2013) The stringency of start codon selection in the filamentous fungus Neurospora crass. J. Biol. Chem. 288, 9549–62.
  3. Paek Ki-Y. et al. (2012) Cap-Dependent translation without base-by-base scanning of an messenger ribonucleic acid. Nucl. Acid. Res. 40, 7541–51.
  4. Se, and NH. Su.W. et al. (2011) Translation, stability, and resistance to decapping of mRNA containing caps substituted in the triphosphate with BH3. RNA 17, 978–88.
  5. Anderson, D. et al. (2011) Nucleoside modifications in RNA limit activation of 2’-5’ oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucl. Acid. Res. 39, 9329-38.

RNA virus Characterization

  1. Vashist, S. et al. (2012) Identification of RNA-protein interaction networks involved in the Norovirus life cycle. J. Vir. 86, 11977–90.
  2. Soto-Rifo, R. et al. (2012) Different effects of the TAR structure on HIV-1 and HIV-2 genomics RNA translation. Nucl. Acids. Res. 40, 2653–67.
  3. Poyry, T. et al. (2011) Mechanisms governing the selection of translation initiation sites on Foot-and-Mouth Disease Virus RNA. J.Vir. 85, 10178–88.
  4. Cheng, E. et al. (2011) Characterization of the interaction between Hantavirus nucleopcapsid protein and ribosomal protein S19. J. Biol. Chem. 286, 11814–24.
  5. Vera-Otarola, J. et al. (2011) The Andes Hantavirus NSs Protein is expressed from the Viral mRMA by a leaky scanning mechanism. J. Vir. 86, 2176–87.