1-Step Human High-Yield In Vitro Translation

The Thermo Scientific 1-Step Human High-Yield In Vitro Translation (IVT) Kits with continuous-feed device enables the expression of functional proteins with 10 to 100 times greater yield per mL than other eukaryotic IVT translation systems.

The 1-Step High-Yield IVT System uses modified HeLa cell extracts to take advantage of the robust human transcription and translation machinery and generate functional full-length proteins. In this system, protein expression is performed in a proprietary mini dialysis device that allows a continuous supply of nucleotides, amino acids and energy generating substrates into the reaction while removing inhibitors of proteins synthesis. This continuous-exchange cell-free (CECF) system enables protein expression for up to 24 hours and produces protein yields ranging from 250 to 750µg/mL. The complete kits come with all components required for proper transcription and translation of recombinant protein, including an optimized expression vector.

Highlights:

• High expression – up to 750µg/mL of expressed protein
• Reproducibility – low variability between experiments
• Fast – express high levels of protein with an overnight incubation
• Functional – obtain more active protein than with bacterial protein expression systems

1-Step Protein Expression:

• Streamlined reaction – requires only 5 components
• Easy-to-use – transcription and translation is performed in one reaction step
• Adapt for labeling – amenable to incorporation of heavy or unnatural amino acids

Includes: Complete kits contain cell lysate, reaction mix, accessory proteins, 5X dialysis buffer, control DNA, cloning vector, mini dialysis devices and centrifuge tubes

Requires: 30°C incubator or water bath

Applications:

• Production of recombinant proteins for structural analysis
• Generate active proteins for protein interaction studies
• Perform rapid expression and characterization of mutant proteins
• Small-scale production of virual particles
• Incorporation of unnatural amino acids or toxic protein production

Product Details:

Procedure overview for continuous-exchange cell-free expression using the Thermo Scientific 1-Step Human High-Yield Maxi IVT Kit. Using the supplied mini dialysis device and buffers, protein expression can be performed overnight at 30°C to generate up to 750µg/mL of protein. Note: For smaller scale and screening options, consider the 1-Step Human Coupled IVT Kit - DNA or mRNA.

The 1-Step Human High-Yield IVT Kits are cell-free protein expression systems that provide all of the essential components required for protein transcription and translation. The kits are optimized with Accessory Proteins and Reaction Mixes that support protein synthesis for up to 24 hours using a DNA template. The advantages of using the 1-Step Human High-Yield IVT Kits over traditional in vivo expression systems include the ability to express toxic or insoluble proteins, easily perform protein labeling with modified amino-acids and reduce the time and cost of expressing human proteins in tissue culture cells. 1-Step Human IVT Kits are optimized for use with the pT7CFE1 expression vector that utilizes the T7 viral promoter and an EMCV Internal Ribosome Entry Site (IRES) to facilitate high levels of in vitro protein expression. Using a pT7CFE vector is critical for obtaining high expression levels. For added convenience, at family of pT7CFE vectors are available with tandem affinity tags for facilitate protein detection and purification.

In vitro expression of Turbo-GFP and p53 using the Thermo Scientific 1-Step Human High Yield IVT Kit. Recombinant p53 and GFP proteins were expressed using the 1-Step Human High-Yield Mini-IVT Kit. At each time point, 5µL aliquots were withdrawn from the reactions and stored at -20°C until assayed. The amount of p53 was quantified by ELISA and Turbo-GFP was quantified using a fluorescent plate-based assay.

Product References:

1. Festa F. et al. (2012) Robust microarray production of freshly expressed proteins in a human milieu. Proteomics Clin Appl. [Epub ahead of print]. Published by Joshua LaBaer's group.
2. Wang J. et al. (2012) A versatile protein microarray platform enabling antibody profiling against denatured proteins. Proteomics Clin Appl. [Epub ahead of print].
3. Boohaker R. J. et al. (2011) BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol. 300, C1466-78.
4. Loughran G. et al. (2011) Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome. Proc Natl Acad Sci U S A. 108, E1111-9.
5. Stergachis, A. (2011). Rapid empirical discovery of optimal peptides for targeted proteomics. Nature Methods, 8: 1041-1043.
6. Wang Q. Y. et al. (2011) A translation inhibitor that suppresses dengue virus in vitro and in vivo. Antimicrob Agents Chemother. 55, 4072-80.
7. Boyne, J. (2010). Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs. EMBO Journal, 29: 1851-1864.
8. Kasinathan, R., (2010). Schistosoma mansoni express higher levels of multidrug resistance-associated protein 1 (SmMRP1) in juvenile worms and in response to praziquantel. Molecular and Biochemical Parasitology, 173: 25-31.
9. Khatua, A. (2010). Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F. Virology, 400: 68-75.

General References:

1. Imataka, H., and Mikami, S. (2009). Advantages of human cell-derived cell-free protein synthesis systems (Japanese). Seikagaku 81(4):303-7.
2. Kobayashi, T., et al. (2007). An improved cell-free system for picornavirus synthesis. J. Virol. Methods 142(1-2):182-8.
3. Kozak, M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361:13-37.
4. Kozak, M. (1983). Comparison of initiation of protein synthesis in prokaryotes, eukaryotes, and organelles. Microbiol. Rev. 47(1):1-45.
5. Mikami, S., et al. (2006). An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr. Purif. 46(2):348-57.

Related Resources:

Getting Started Guide – overview of critical parameters for successful human in vitro protein expression
Review of cell-free in vitro recombinant protein expression

Related Products:

pT7CFE1 Expression Vectors
Custom Expression Vector Construction Service
1-Step Human IVT Kits for DNA or mRNA
1-Step Heavy Protein IVT Kits