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.
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.
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.
Protein phosphorylation is one of the most biologically relevant modifications and is involved in many eukaryotic and prokaryotic cellular signaling processes. It is estimated that one-third of human proteins are phosphorylated.
The following examples utilize the ability of cell free experession to express active proteins, and when supplemented with the necessary components (e.g., ATP, NaCl), to be used for the characterization of phosphorylation events.
mRNAs commonly exhibit differing salt requirements for optimal translation. Small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi® Rabbit Reticulocyte Lysate System allows translation reactions to be optimized for a wide range of parameters, including
Mg2+ and K+ concentrations and the choice of adding DTT. To help optimize Mg2+ for a specific message, the endogenous Mg2+ concentration of each lysate batch is stated in the product information included with this product.
The following references utilize the features of Flexi Rabbit Reticulocyte Lysate System to investigate certain parameters of translation:
Precise mapping of the positions of ribosomes and associated factors on mRNAs is essential for characterizing the mechanism of translation. Using the toeprinting assay, mRNA is translated using purified components or crude cell lysates such as rabbit reticulocyte. Cycloheximide is added to the reaction to inhibit elongation. This arrests the position of the ribosomes on the mRNA transcript. The mRNA/ribosomal complex are then copied into cDNA by reverse transcriptase using a complementary radiolabeled primer. Where the reverse transcriptase meets the ribosome bound to the mRNA, cDNA extension is halted, and a toeprint cDNA fragment is generated.
The following references use rabbit reticulocyte lysates as the basis for toeprinting experiments to better understand the mechanism of translation.
Weill, L. et al. (2010)Nucl. Acid, Res. 38, 1367–81. A combination of chemical/enzymatic analyses indicated that gag open reading frame of three viruses adopts a stable secondary structure that allows IRES mediated translation. Mutations that destabilized conserved elements severely inhibit translation. Additional analysis via toeprinting showed HIV-2 IRES has the unique ability to attract up to three initiation complexes on a single RNA molecule.
De Breyne, S. et al. (2008) RNA14, 367–80. The Simian picornavirus type 9 (SPV9) genome contains a group of IRES that resembles hepacivirus/pestvirus (HP) IRES. Characterization of the initiation process using the toeprinting assay in correlation with other techniques revealed aspects that resemble initiation on the HP IRES and others that are unique to SPV9.
Andreev, D. et al. (2008) RNA14, 233–39. Rel E is a well characterized toxin involved in the nutritional stress response in bacteria and archae. Rel lacks any eukaryote homolog. Based on toeprinting data, it was demonstrated that RelE cleaves mRNA in the A site of the eukaryote ribosome.
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.
Many proteases have proven useful for monitoring translocation in this fashion including Protease K or Trypsin.
The following are examples illustrating this application:
Cell free protein expression has become a convenient method for generating small amounts of protein for a variety of applications (e.g. protein:protein interactions, protein: nucleic acid interactions). One of the most popular formats is based on rabbit reticulocytes. Using a mammalian based system offers researchers the optimal environment for the expression of functional proteins (e.g. correctly folded or modified).
When using a rabbit reticulocyte based translation system to express your protein of interest followed by detection using Western blotting there are a few critical parameters to keep in mind in order to optimize your experimental data. Excessive background can be caused by several factors.
One of the most common mistakes is loading too much lysate on the gel. Typically loading between 0.5µl and 2µl of lysate should provide an adequate signal to detect.
Be sure not to use a secondary antibody directed against rabbit proteins, since it will react with the lysate components.
If you are experiencing background when using horseradish peroxidase detection, the peroxidase activity from the hemoglobin maybe the issue. To prevent the hemoglobin from reacting with the substrate during detection, soak the membrane in TBS containing 10% ImmunoPure Peroxidase Suppressor (Pierce Cat # 35000) for 30 minutes prior to blocking.
Cell-free protein synthesis has emerged as powerful alternative to cell based protein expression for functional and structural proteomics. The TNT® SP6 High-Yield Protein Expression System uses a high-yield wheat germ extract supplemented with SP6 RNA polymerase and other components. Coupling transcriptionaland translational activities eliminates the inconvenience of separate in vitro transcription and purification steps for the mRNA, while maintaining the high levels of protein expression (1). Continue reading “Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein”
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: