Thermo Scientific 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100µg/mL per reaction.
The Human IVT Kits are unique HeLa cell lysate-based protein expression systems for in vitro translation or coupled transcription/translation reactions. Protein expression is performed in a single 90-minute reaction that can be extended for up to 6 hours with continued protein production up to 100µg/mL when combined with the optimized pT7CFE1 Expression Vector. The Human IVT Kits can express functional enzymes, phosphoproteins, glycoproteins and membrane proteins for immediate use in studying protein interactions, performing rapid mutational analysis and measuring activity.
Highlights:
- Functional – uses the human translational machinery to express active proteins
- Convenient – perform transcription and translation in a single step
- High performance – greater yields compared to rabbit reticulocyte in vitro translation
- Reliable – express proteins that fail in rabbit reticulocyte systems
Better Than Traditional Methods:
- HeLa cell-free extracts are capable of expressing proteins with post-translational modifications
- Accurate translation delivers full-length protein compatible with downstream applications
- Protein translation is optimized with EMCV IRES element and other mRNA stabilizing elements
Includes: Complete kits contain HeLa cell lysate, accessory proteins, reaction mix, nuclease-free water, expression vector and a GFP-positive control vector
Requires: 30°C incubator or water bath
Applications:
- Express proteins to measure enzyme activity
- Perform mutational analysis
- Express protein for use in gel mobility shift assays
- Perform co-immunoprecipitation
- Express cytotoxic proteins
- Perform unnatural amino acid labeling
Product Details:
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Schematic of the 1-Step Coupled Human IVT Kit for DNA and 1-Step Human IVT Kit for RNA. Simply add the appropriate template to a mixture of HeLa cell lysate, Accessory Proteins, Reaction Mix and incubate at 30°C for 90 minutes for protein yeilds up to 100µg/mL. Smaller reactions are ideal for expression of mutational variants in a microplate format. The reaction volumes and times can be increased to express larger amounts of a single protein for use in several downstream applications.
Note: For even more protein expression, 250 to 750µg/mL of protein can be achieved using the 1-Step High-Yield IVT Kits. |
The 1-Step Coupled Human IVT Kit for DNA is a cell-free system using the cellular transcription and translation machinery from a modified HeLa cell line. The procedure is quick and easy and is an effective alternative to other protein expression methods. Simply add an appropriate expression construct to a mixture of HeLa cell lysate, Accessory Protiens, Reaction Mix and incubate at 30°C for 90 minutes to overnight.
The 1-Step Human IVT Kit for mRNA also uses the human protein translation machinery to produce functional protein. This kit is recommended for translation of mRNA transcripts containing an EMCV IRES element and mRNA stabilizing features designed into the pT7CFE1 expression vector.
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| The Thermo Scientific 1-Step Human Coupled Human IVT Kit produces more active protein without interfering substances. In vitro luciferase expression reactions were performed with the 1-Step Human Coupled IVT Kit and the Promega TnT* T7 Quick Coupled Transcription/Translation System according to supplied instructions and control plasmids. Samples were removed from each reaction at the indicated intervals and analyzed for (A.) luciferase activity (correlated to µg/mL of active protein) or ( B.) Western blot (1µL). The 1-Step Coupled Human In Vitro Expression Kit produced luciferase protein without contaminating beta-globin. |
The human in vitro translation system is robust and will express proteins from a variety of species including mammals, bacteria and protozoa. Benchmarking shows that the expression levels of functional proteins such as luciferase are much higher in the human system compared to either rabbit reticulocyte-based or E. coli-based systems. Furthermore, proteins expressed with the human in vitro translation system are not contaminated with substances that can interfere with downstream applications.
Product References:
- 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.
- Wang J. et al. (2012) A versatile protein microarray platform enabling antibody profiling against denatured proteins. Proteomics Clin Appl. [Epub ahead of print].
- Yadavalli, R., Ledger, C., Sam-Yellowe, TY. (2012). In vitro human cell-free expression system for synthesis of malaria proteins. Parasitol Res. 2012 Jul 11. [Epub ahead of print] DOl I 0.1007 /s00436-0 12-3014-7.
- Boohaker R. J. et al. (2011). BAX supports the mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J Physiol Cell Physiol. 300, C1466-78.
- 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.
- Stergachis, A. (2011). Rapid empirical discovery of optimal peptides for targeted proteomics. Nature Methods, 8: 1041-1043.
- Wang Q. Y. et al. (2011). A translation inhibitor that suppresses dengue virus in vitro and in vivo. Antimicrob Agents Chemother. 55, 4072-80.
- 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.
- 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.
- Khatua, A. (2010). Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F. Virology, 400: 68-75.
General References:
- Imataka, H., and Mikami, S. (2009). Advantages of human cell-derived cell-free protein synthesis systems (Japanese). Seikagaku 81(4):303-7.
- Kobayashi, T., et al. (2007). An improved cell-free system for picornavirus synthesis. J. Virol. Methods 142(1-2):182-8.
- Kozak, M. (2005). Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361:13-37.
- Kozak, M. (1983). Comparison of initiation of protein synthesis in prokaryotes, eukaryotes, and organelles. Microbiol. Rev. 47(1):1-45.
- 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 High-Yield In Vitro Protein Expression Kits
1-Step Heavy Protein IVT Kits
Anti-Turbo GFP Polyclonal Antibody (Part No. PA5-22688)
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