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1-Step Human High-Yield In Vitro Translation Kits


Express up to 0.75mg per mL of functional protein in less than 16 hours.

1-Step Human High-Yield In Vitro Translation Kits

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

The 1-Step High-Yield IVT System uses modified HeLa cell extracts to take advantage of the robust human translation machinery and generate functional full-length proteins. In this system, protein expression is performed in a proprietary 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 in an overnight incubation of up to 750µg/mL. The complete mini- and maxi-scale kits include all the components required for transcription and translation of a recombinant gene, 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 6 hours to overnight incubation
  • Functional – obtain functionally active proteins, including those containing disulfide bonds

1-Step Protein Expression:

  • 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, dialysis devices and centrifuge tubes

Requires:

  • 30°C incubator or water bath (shaker incubator increases expression by 30 to 50%)

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 viral particles
  • Incorporation of unnatural amino acids or toxic protein production

Product Details:

Protocol summary for cell-free protein expression with the 1-Step Human High-Yield Maxi IVT Kit.
Procedure summary for the Maxi IVT kit. (Click image to view larger.) The reaction is performed in a dialysis device inserted into a 50mL conical tube. The first five steps are for equilibration and preparation of the dialysis device. The IVT reaction mixture is added in step 6.

 

Protocol summary for cell-free protein expression with the 1-Step Human High-Yield Mini IVT Kit.
Procedure summary for the Mini IVT kit. (Click image to view larger.) The reaction is performed in a microdialysis device inserted into a 2mL microcentrifuge tube.

The 1-Step Human High-Yield IVT Kits are cell-free protein expression systems that provide all of the essential components required for transcription and translation. The kits are optimized with Accessory Proteins and Reaction Mixes that support protein synthesis 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. (For smaller scale and screening options, consider the 1-Step Human Coupled IVT Kit.)

High-yield in vitro protein expression and purification of GST-tagged proteins
Purification of GST fusion proteins expressed by the 1-Step Human High-Yield IVT Kit. Proteins were purified using glutathione agarose with elution by either 10mM glutathione or HRV3C protease cleavage, respectively. In the lanes for Bad protein, the additional bands (triangle) are 14-3-3 proteins, which co-elute with Bad. Protein identification was verified by mass spectrometry (data not shown). These purification strategies are described in greater detail in a related article.

 

High-yield in vitro protein expression of GST-tagged proteins

Expression of coomassie-stainable levels of proteins using the 1-Step Human High-Yield IVT Kit. Five expression-ready clones (pANT7-based vector) obtained from the DNASU Plasmid Repository were used to express the GST-fusion proteins listed in Lanes 3-7. Lane 2 shows expression of the control pCFE-GFP plasmid.  Reaction mixtures of 5µL were separated by SDS-PAGE and stained with GelCode Blue Stain Reagent (Part No. 24590). Arrows indicate the positions of expressed proteins.

 

Protein yields for each Thermo Scientific 1-Step Human High-Yield IVT Kit. Expected yield is for the positve control GFP protein (vector included in each kit).
Kit Size(s) Reaction
Volume
Expected
Yield
1-Step Human High-Yield Mini IVT Kit 2-rxn and 10-rxn kits 0.1mL 0.25mg/mL
1-Step Human High-Yield Maxi IVT Kit 2-rxn kit 2mL 0.25mg/mL

 

More Product Data

Product References:

  1. Heidary DK, Glazer EC.(2014). A light-activated metal complex targets both DNA and RNA in a fluorescent in Vitro transcription and translation assay. Chembiochem. 15(4):507-11.
  2. Festa F., et al. (2013) Robust microarray production of freshly expressed proteins in a human milieu. Proteomics Clin Appl. 7(5-6):372-7. Published by Joshua LaBaer's group.
  3. Wang J., et al. (2013) A versatile protein microarray platform enabling antibody profiling against denatured proteins. Proteomics Clin Appl. 7(5-6):378-83.
  4. Boohaker R. J. et al. (2011) BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol. 300, C1466-78.
  5. Loughran G. et al. (2011) Ribosomal frameshifting into an overlapping gene in the 2B-encoding region of the cardiovirus genome. Proc Natl Acad Sci USA. 108, E1111-9.
  6. Stergachis, A. (2011). Rapid empirical discovery of optimal peptides for targeted proteomics. Nature Methods, 8:1041-43.
  7. Wang Q. Y. et al. (2011) A translation inhibitor that suppresses dengue virus in vitro and in vivo. Antimicrob Agents Chemother. 55:4072-80.
  8. Boyne, J. (2010). Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs. EMBO Journal, 29:1851-64.
  9. 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.
  10. 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


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
88890 1-Step Human High-Yield Mini IVT Kit
Formulation: Multi-component kit
Sufficient For: 2 reactions of 100μL each

Kit Contents:
HeLa Lysate, 110μL
Accessory Proteins, 25μL
Reaction Mix, 40μL
5X Dialysis Buffer, 600μL
Positive Control DNA (pCFE-GFP), 10μg
pT7CFE1-NHis-GST-CHA, 10μg
Microdialysis Device, 2 each
Nuclease-free Water, 5mL
2 x 100µL kit Product Instructions for product #88890 1-Step Human High-Yield Mini IVT Kit MSDS for product #88890 1-Step Human High-Yield Mini IVT Kit $235.00

Add to cart
88891 1-Step Human High-Yield Mini IVT Kit
Formulation: Multi-component kit
Sufficient For: 10 reactions of 100μL each

Kit Contents:
HeLa Lysate, 500μL
Accessory Proteins, 100μL
Reaction Mix, 200μL
5X Dialysis Buffer, 3mL
Positive Control DNA (pCFE-GFP), 10μg
pT7CFE1-NHis-GST-CHA, 10μg
Microdialysis Device, 10 each
Nuclease-free Water, 3 x 5mL
10 x 100µL kit Product Instructions for product #88891 1-Step Human High-Yield Mini IVT Kit MSDS for product #88891 1-Step Human High-Yield Mini IVT Kit $1,135.00

Add to cart
88892 1-Step Human High-Yield Maxi IVT Kit
Formulation: Multi-component kit
Sufficient For: 2 reactions of 2mL each

Kit Contents:
HeLa Lysate, 4 x 500μL
Accessory Proteins, 4 x 100μL
Reaction Mix, 4 x 200μL
5X Dialysis Buffer, 23mL
Positive Control DNA (pCFE-GFP), 10μg
pT7CFE1-NHis-GST-CHA, 10μg
Maxi dialysis Device, 2 each
Nuclease-free Water, 3 x 50mL
2 x 2mL kit Product Instructions for product #88892 1-Step Human High-Yield Maxi IVT Kit MSDS for product #88892 1-Step Human High-Yield Maxi IVT Kit $3,200.00

Add to cart

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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