Author: Gary Kobs

Using Pepsin to Prepare F(ab’)2 Fragments and Determine Deuterium Exchange

Posted on by Gary Kobs

Using Pepsin to Prepare F(ab’)2 Fragments

Pepsin is commonly used in the preparation of F(ab’)2 fragments from antibodies. In some assays, it is preferable to use only the antigen-binding (Fab) portion of the antibody. For these applications, antibodies may be enzymatically digested to produce an F(ab’)2 fragment of the antibody. To produce an F(ab’)2 fragment, IgG is digested with pepsin, which cleaves the heavy chains near the hinge region. One or more of the disulfide bonds that join the heavy chains in the hinge region are preserved, so the two Fab regions of the antibody remain joined together, yielding a divalent molecule (containing two antibody binding sites), hence the designation F(ab’)2. The light chains remain intact and attached to the heavy chain. The Fc fragment is digested into small peptides.

Continue reading “Using Pepsin to Prepare F(ab’)2 Fragments and Determine Deuterium Exchange”

High-Yield Cell-Free Protein Expression: Prokaryotic Based

Posted on by Gary Kobs

S30 E coli high yield extract schematicMany applications require amounts of protein that cannot be obtained using a eukaryotic cell-free expression system. As an alternative, a prokaryotic system can be used when this need arises. The E. coli S30 T7 High-Yield Protein Expression System is designed to express up to 500μg/ml of protein in 1 hour from plasmid vectors containing a T7 promoter and a ribosome binding site. The protein expression system provides an extract that contains T7 RNA polymerase for transcription and is deficient in OmpT endoproteinase and lon protease activity. All other necessary components in the system are optimized for protein expression. This results in greater stability and enhanced expression of target proteins.The following references highlight the use of this system for a variety of unique applications:

Loh, E. et al. (2011) An unstructured 5′-coding region of the prfA mRNA is required for efficient translation. Nuc. Acids. Res. (online) Examines the effect of upstream codon sequence/length on the correct ribosome binding and translation initiation of the pfrA protein.

Mitsuhashi, H. et al. (2010) Specific phosphorylation of Ser458 of A-type lamins in LMNA-associated myopathy patients. J. Cell. Sci. 123, 3893–900 By creating a series of mutations in the protein lamin A, Akt1 phosphorylation sites were determined.

Halvorsen, E. et al. (2011) Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis. Microbiology 157, 387–97. S30 High Yield System was used to characterize the inhibitory effect of Txe toxin on protein expression.

Mo, P. et al. (2010) MDM2 mediates ubiquitination and degradation of activating transcription factor 3. J. Biol. Chem. 285, 26908–15. By using in vitro pull down experiments the researchers characterized the binding of AFT3 to MDM2 leading to the proteolysis of AFT3 system by ubiquitination.

Use of Nonspecific Proteases for Analysis of Proteins by Mass Spectrometry

Posted on by Gary Kobs
mass spectrometry results

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.

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Nonspecific proteases such as pepsin, proteinase K, elastase and thermolysin can offer an alternative to traditional sequence-specific proteases for certain applications. The following references illustrate the use of nonspecific proteinases for the mass spec analysis of proteins:

Papasotiriou, D. et al. (2010) Peptide mass fingerprinting after less specific in-gel proteolysis using MALDI-LTQ-Orbitrap and 4-chloro-alpha-cyanocinnamic acid. J. Proteome. Res. 9, 2619–29. This reference demonstrates the use of either chymotrypsin, elastase, trypsin or proteinase K in combination with matrix CHCA for increase peptide identification and sequence coverage using MALDI.

Neue, K. et al. (2011) Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides. J. Proteome. Res. 10, 2248–60. Notes use of thermolysin or elastase in combination with ZIC-HILIC enrichment as alternative method for the characterization of glycopeptides.

Baeumlisberger, D et al. (2011) Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes. J. Proteome. Res. 10, 2889–94. Data noted that samples digested with elastase followed by nLC separation and subsequent alternative spotting on both MALDI-LTQ-Orbitrap and MALDL-TOF/TOF instruments resulted in 32% additional peptides.

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 Kinase Assays

Posted on by Gary Kobs

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.

Modrof, J. et al. (2005) Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies. J. Vir. 79, 10023–31. Use of TNT® cell-free to express NS2 and NS2 mutant proteins for use in vitro kinase assays to confirm phosphorylation by protein kinase CK2.

Kwon, S. et al. (2005) Signal pathway of hypoxia-inducible factor-1alpha phosphorylation and its interaction with von Hippel-Lindau tumor suppressor protein during ischemia in MiaPaCa-2 pancreatic cancer cells. Clin. Cancer Res. 11, 7607–13. The TNT® system was used to identify which p38 mitogen-activated protein kinase isoform(s) was cabable of phosphorylation of HIF—1 alpha

Harris, J. et al. (2006). Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon. J. Immunol. 177, 2527–35. IKK and IKK mutants were expressed using TNT and used in a vitro kinase assay to characterize the recognition motif in cRel transcription domain

Jailais, Y. et al. (2011) Tyrosine phosphorylation controls brassinosteroid receptor activation by triggering membrane release of its kinase inhibitor. Genes Dev. 25, 232–37. Using a vitro kinase assay, full–length and truncations versions of the Brassinostediod-insentive receptor protein were expressed using the TNT® system and incubated with purified BR11 kinase domain to determine binding sites of the two proteins.

Optimized Protein Expression: Flexi Rabbit Reticulocyte Lysate

Posted on by Gary Kobs
A protein chain being produced from a ribosome.

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:

Vallejos, M. et al. (2010)The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation. Nucl Acids Res. 38, 618–32. Identification of internal ribosomal ribosomal entry site in the 5’ untranslated region of the mouse mammary tumor virus mRNA.

Spriggs, K. et al. (2009) The human insulin receptor mRNA contains a functional internal ribosome entry segment. Nucl. Acids. Res. 17, 5881–93. Identification of a functional internal ribosome entry site in the human insulin receptor mRNA.

Powell, M. et al. (2008) Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein. RNA 14, 2394–06. Analysis of the mRNA signals involved in the expression of influenza B virus BM2 protein.

Sato, V. et al. (2007) Measles virus N protein inhibits host translation by binding to eIF3-p40. J. Vir. 81, 11569–76. Charaterized the effect of the measles virus N protein binding to the translation initiation factor eIF3-p40 on the expression of various proteins in rabbit reticulocyte lysate.

Hirao, K. et al. (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J. Biol. Chem. 281, 9650–58. The EDEM3 protein was expressed in the presence of canine microsomal membranes to establish that co-translational translocation occurs into the endoplasmic reticulum.

Shenvi, C. et al. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations–implications for the study of ribosome dynamics. RNA 11, 1898–08. Characterization of ribosome dynamics under different ionic conditions.

Nair, A. et al. (2005) Regulation of luteinizing hormone receptor expression: evidence of translational suppression in vitro by a hormonally regulated mRNA-binding protein and its endogenous association with luteinizing hormone receptor mRNA in the ovary. J. Biol. Chem. 280, 42809–16. Examined the affect of luteinizing hormone receptor mRNA binding protein on transltional suppression of luteinizing hormone receptor RNA.

Optimization of Western Blots Detecting Proteins Synthesized Using Cell-Free Expression #2

Posted on by Gary Kobs

Detection of protein expressed using cell-free systems is required for most applications such as protein:protein interaction and protein:nucleic acid interaction studies. Traditionally, one adds radioactive [35S]methionine to cell-free expression reactions, and the methionine is incorporated into the expressed protein, allowing detection by autoradiography. Many researchers are moving away from radioactivity. Traditional Western blot analysis provides the researcher a nonradioactive method for detection but, if performed improperly, can result in high background, which can mask expressed proteins and affect downstream applications.

One critical step in producing low-background, high-signal Western blots is choosing the correct dilution of the primary antibody. Typically the manufacturer recommends antibody dilution from 1:1,000 to 1:2,500 for standard western blotting experiments. However when using crude lysates as a source of the target protein, these recommendations exhibit significant background. When the antibody was diluted 1:50,000, background was decreased significantly, and the positive signal was a large percentage of the total signal.

As a general recommendation when performing Western blot analysis of proteins expressed in cell-free systems, one must experimentally determine the optimal dilution of the primary antibody. In the Western blots performed in this study, primary antibodies were diluted ~50-fold more than the provider’s recommended dilution.

For additional technical details refer to this recent article published in Promega’s PubHub:

Hook, B and Schagat, T. (2011) Non-Radioactive Detection of Proteins Expressed in Cell-Free Expression Systems Promega Corporation Web site. Accessed August 17, 2011.

Cell-Free Applications: RNA Toeprinting

Posted on by Gary Kobs

A protein chain being produced from a ribosome.
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) RNA 14, 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) RNA 14, 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.

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”

Cell-Free Applications:Protein Arrays (Nucleic Acid Programmable)

Posted on by Gary Kobs

The traditional methods of generating protein arrays require the separate expression of hundreds of proteins, followed by purification and immobilization of the proteins on a solid surface. Cell-Free protein array technology produces protein microarrays by performing in vitro synthesis of the target protein from their DNA templates.
One approach for the generation of cell- free based microarrays is the nucleic acid programmable protein array (NAPPA).

NAPPA uses DNA template that is biotinylated and is bound to avidin that is pre-coated onto the protein capture surface. Newly synthesized proteins which are tagged with GST are then immobilized next to the template DNA by binding to an adjacent polyclonal anti-GST capture antibody. The following references illustrate the use of NAPPA to screen hundreds of proteins. Continue reading “Cell-Free Applications:Protein Arrays (Nucleic Acid Programmable)”