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.
- 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
- 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
|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.
|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.
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Getting Started Guide – overview of critical parameters for successful human in vitro protein expression
Review of cell-free in vitro recombinant protein expression
pT7CFE1 Expression Vectors
Custom Expression Vector Construction Service
1-Step Human High-Yield In Vitro Protein Expression Kits
1-Step Heavy Protein IVT Kits
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