Tips and Tools

Screening for Protein Activity Using Cell-Free Expression

Posted on by Gary Kobs

The analysis of functional protein typically requires lengthy laborious cell based protein expression that can be complicated by the lack of stability or solubility of the purified protein. Cell free protein expression eliminates the requirement for cell culture thus providing quick access to the protein of interest (1).

The HaloTag® Technology provides efficient, covalent and oriented protein immobilization of the fusion protein to solid surfaces (2).

A recent publication demonstrated the feasibility of using cell free expression and the HaloTag technology to express and capture a fusion protein for the rapid screening of protein kinase activity (3). The catalytic subunit of human cAMP dependent protein kinase was expressed in a variety of cell free expression formats as a HaloTag fusion protein. The immobilized cPKA fusion protein was assayed directly on magnetic beads in the active form and was shown to be inhibited by known PKA inhibitory compounds.

Therefore this unique combination of protein expression and capture technologies can greatly facilitate the process of activity screening and characterization of potential inhibitors

References
ResearchBlogging.org

  1. Zhao, K.Q. et al. (2007) Functional protein expression from a DNA based wheat germ cell-free system. J. Struc. Funct. Genomics. 8, 199-208.
  2. Los, G.V. and Wood, K. (2007) The HaloTag: A novel technology for cell imaging and protein analysis. Meth. Mol. Biol. 356, 195-208
  3. Leippe DM, Zhao KQ, Hsiao K, & Slater MR (2010). Cell-free expression of protein kinase a for rapid activity assays. Analytical chemistry insights, 5, 25-36 PMID: 20520741

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.

6X His Protein Pulldowns: An Alternative to GST

Posted on by Gary Kobs

ResearchBlogging.orgPull-down assays probe interactions between a protein of interest that is expressed as fusion protein (e.g.,
(e.g., bait) and the potential interacting partners (prey).

In a pull-down assay one protein partner is expressed as a fusion protein (e.g., bait protein) in E. coli and then immobilized using an affinity ligand specific for the fusion tag. The immobilized
bait protein can then be incubated with the prey protein. The source of the prey protein depends on whether the experiment is designed to confirm an interaction or to identify new interactions. After a series of wash steps, the entire complex can be eluted from the affinity support using SDS-PAGE loading buffer or by competitive analyte elution, then evaluated by SDS-PAGE.

Successful interactions can be detected by Western blotting with specific antibodies to both the prey and bait proteins, or measurement of radioactivity from a [35S] prey protein. bait) and potential interacting partners (prey).

The most commonly used method to generate a bait protein is expression as a fusion protein contain a GST (glutathione-S transferase) tag in E. coli. This is followed by immobilization on particles that contain reduced glutathione, which binds to the GST tag of the fusion protein. The primary advantage of a GST tag is that it can increase the solubility of insoluble or semi-soluble proteins expressed in E. coli.

Among fusion tags, His-tag is the most widely used and has several advantages including: 1) It’s small in size, which renders it less immunogenically active, and often it does not need to be removed from the purified protein for downstream applications; 2) There are a large number of commercial vectors available for expressing His-tagged proteins; 3) The tag may be placed at either the N or C terminus; 4) The interaction of the His-tag does not depend on the tag structure, making it possible to purify otherwise insoluble proteins using denaturing conditions. Continue reading “6X His Protein Pulldowns: An Alternative to GST”

Use of Multiple Proteases for Improved Protein Digestion

Posted on by Gary Kobs

One of the approaches to identify proteins by mass spectrometry includes the separation of proteins by gel electrophoresis or liquid chromatography. Subsequently the proteins are cleaved with sequence-specific endoproteases. Following digestion the generated peptides are investigated by determination of molecular masses or specific sequence. For protein identification the experimentally obtained masses/sequences are compared with theoretical masses/sequences compiled in various databases.

Trypsin is the favored enzyme for this application, for the following reasons: A) the peptides contain a basic residue (Arg or Lys) on the C terminus and thus are good candidates for collision induced activation (CAD) in tandem experiments (low charge states and high mass-to-charge ratios); B) it is relatively Inexpensive; and C) optimal digestion conditions have been well characterized.

An inherent limitation of trypsin is the size of the peptides that it generates. For most organisms > 50% of tryptic peptides are less than 6 amino acids, too small for mass spectrometry based sequencing.

Are you looking for proteases to use in your research?
Explore our portfolio of proteases today.

One recent publication examined the use of multiple proteases (trypsin, LysC, ArgC , AspN and GluC) in combination with either CAD or electron-based fragmentation (ETD) to improve protein identification (1). Their results indicated a significant improvement from a single protease digestion (trypsin), which yielded 27,822 unique peptides corresponding to 3313 proteins. In contrast using a combination of proteases with either CAD or ETD fragmentation methods yielded 92,095 unique peptides mapping to 3908 proteins.

Swaney DL, Wenger CD, & Coon JJ (2010). Value of using multiple proteases for large-scale mass spectrometry-based proteomics. Journal of proteome research, 9 (3), 1323-9 PMID: 20113005

Trypsin: Innovative Applications

Posted on by Gary Kobs
3D model of protein and protease cleavage

Tryptic digestion of samples and subsequent analysis by mass spectrometry is a popular technique for the identification of proteins typically those related to interaction partners or biomarkers characterization. This powerful tool can also be used for less traditional experimental designs. Three examples are:

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Humorous New Types of PCR

Posted on by Terri Sundquist

Undoubtedly, the polymerase chain reaction (PCR) has revolutionized biological research and has become one of the most common techniques in today’s laboratory. At times, it seems that a new variation of PCR is described in the literature every month. You might think that you are familiar with the dozens of PCR variations, but I am guessing that you haven’t heard of some of these.

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Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein

Posted on by Gary Kobs
Wheat Germ Extract for high-yield protein expression

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).

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Variations on the Two-Hybrid Assay

Posted on by Isobel Maciver
two-hybrid assays help fit molecules together like puzzle pieces image shows a puzzle

The use of reporter genes for simple analysis of promoter activity (promoter bashing) is a well known practice. However, there are many other elegant applications of reporter technologies. One such application is illustrated in the paper by Zheng et al., published in the Sept. 2008 issue of Cancer Research. These researchers from the Hormel Institute at the University of Minnesota showed that the cyclin-dependent kinase cdk3 phosphorylates the transcription factor ATF1 and enhances its transcriptional and transactivation activity. The observed cdk/ATF1 signaling was shown to have an important role in cell proliferation and transformation. To do this they used several variations of a reporter-based two-hybrid assay.

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Describing Life and Death in the Cell

Posted on by Michele Arduengo

4621CALife is complicated. So is death. And when the cells in your multiwell plate die after compound treatment, it’s not enough to know that they died. You need to know how they died: apoptosis or necrosis? Or, have you really just reduced viability, rather than induced death? Is the cytotoxicity you see dose-dependent? If you look earlier during drug treatment of your cells, do you see markers of apoptosis? If you wait longer, do you observe necrosis? If you reduce the dosage of your test compound, is it still cytotoxic? Continue reading “Describing Life and Death in the Cell”

Why Two Reporters are Better than One

Posted on by Isobel Maciver

As part of my job I occasionally search the literature for papers citing use of Promega products in new or interesting ways. Any search on dual-luciferase reporters usually generates a lot of returns. A search for dual-luciferase on Highwire press generates over 700 articles from 2009 alone. So why are dual-luciferase reporter assays so widely used? Continue reading “Why Two Reporters are Better than One”