in vitro protein expression

Cell free application: Sumoylation characterization

Posted on by Gary Kobs

Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are covalently attached to and detached from other proteins in cells to modify their function. SUMOylation is a post-translational modification involved in various cellular processes, such as nuclear-cytosolic transport,
transcriptional regulation, apoptosis, protein stability and response to stress.

In contrast to ubiquitin, SUMO is not used to tag proteins for degradation. Mature SUMO is produced when the last four amino acids of the C-terminus have been cleaved off to allow formation of an isopeptide bond between the C-terminal glycine residue of SUMO and an acceptor lysine on the target protein.

Cell free expression can be used to characterize sumoylation of proteins. Target proteins are expressed in a rabbit reticulocyte cell free system (supplemented with necessary addition components,). Proteins that have been modified can be analyzed by a shift in migration on polyacrylamide gels, when compared to control reactions.

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

Brandl, A. et al. (2012) Dynamically regulated sumoylation of HDAC2 controls p53 deacetylation and restricts apoptosis following genotoxic stress. J. Mol. Cell. Biol. (online only)

Janer, A. et al. (2010). SUMOylation attenuates the aggregation propensity and cellular toxicity of the polyglutamine expanded ataxin-7. Human. Mol. Gen. 19, 181—95.

Rytinki, M. et al. (2009) SUMOylation attenuates the function of PGC-1alpha. J. Biol. Chem. 284, 26184-93.

Klein, U. et al. (2009) RanBP2 and SENP3 function in a mitotic SUMO2/3 conjugation-deconjugation cycle on Borealin. Mol. Cell. Biol. 20, 410–18.

Seo, W. and Ziltener, H. (2009) CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis. Blood, 114, 3567–77.

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)”

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).

Continue reading “Optimized Wheat Germ Extract for High-Yield Protein Expression of Functional, Soluble Protein”