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Human In Vitro Translation Products
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The Thermo Scientific Pierce Human In Vitro Protein Expression System is a method for expressing proteins from DNA or mRNA templates in a cell-free solution containing essential components of the cellular translational machinery. Extracts of an immortalized human cell line provide the ribosomes, initiation and elongation factors, tRNAs and other basic components required for protein synthesis. When supplemented with proprietary accessory proteins, ATP, and an energy regenerating system, these extracts sustain the synthesis of target proteins from DNA templates for up to 6 hours without the need to remove inhibitory byproducts.
The benefits of in vitro protein expression include compatibility with microliter-scale reactions and speed compared to traditional cell-based methods (overnight for bacterial cultures or weeks for baculoviral protein preparations). This small-scale expression method makes it easy to express numerous mutant variants simultaneously in a microplate or express large quantities of a single protein for future experiments. Small-scale synthesis also helps to avoid protein aggregation into inclusion bodies, a typical problem for bacterial expression systems. Additionally, expression of proteins in vitro enables synthesis of toxic proteins that can not be produced in live cells.
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Types of proteins expressed by human in vitro translation |
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| Thermo Scientific Pierce Human In Vitro Translation Kit procedure. Protein synthesis begins with either a DNA or RNA template. When starting with DNA, mRNA is transcribed in one hour at 32°C using the included reaction components. The mRNA are then added to the in vitro translation reaction and incubated for 90 minutes at 30°C for general protein expression. Alternatively, the reaction is incubated for 90 minutes at 28°C for glycoprotein expression. |
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Human In Vitro Protein Expression Kits
The Human In Vitro Protein Expression Kits use human cell lysates to synthesize proteins. Compared to non-mammalian (E. coli) or alternative species (e.g., insect, wheat germ and rabbit reticulocyte lysates) systems, in vitro protein expression using human cell extracts delivers functional proteins within hours. Expression can be achieved using either a DNA or mRNA template, which can be derived from a vector or acquired through our library of gene open reading frames.
The protein expression system can express protein from any species and has been validated with numerous genes from both prokaryotic and eukaryotic sources.
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Pierce Human In Vitro Protein Expression Kits
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Thermo Scientific cDNAs, ORFs, Promoters and other genomics-related products
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In vitroprotein expression in a human system enables easy detection of fluorescent proteins. Translation reactions containing green fluorescent protein (GFP) mRNA were performed with the Thermo Scientific Pierce Human In Vitro System (Human) and a leading rabbit reticulocyte lysate system (Rabbit). Expression of tGFP was easily monitored directly in reaction tubes using a FITC filter. The Pierce System is compatible with fluorescence and colorimetric protein detection. The rabbit reticulocyte lysate system interferes with fluorometric as well as colorimetric detection. |
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Glycoprotein Expression Kits
The major advantage of using the Pierce Human In Vitro Expression System is the ability to generate protein modifications just as they occur in vivo. The Glycoprotein Expression Kits are optimized for generating glycosylated protein. The yield of glycoprotein produced in the system out-performs traditional methods (i.e., those based on rabbit reticulocyte lysates combined with microsomal membranes) and have higher fidelity than insect expression systems, which lack N-linked oligosaccharide side chains.
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| The Thermo Scientific Pierce Human In Vitro Glycoprotein Expression System performs better than insect lysates. Human choriogonadotropin hormone (hCG) β subunit was expressed in insect cell extract or the Pierce System according to the manufacturer's instructions. Insect lysates yielded no glycosylated protein when 1µg of hCGβ mRNA was used and multiple glycoforms when 12µg of mRNA was used. Note that the hCGβ was cloned into vectors that were recommended by the manufacturer of the insect expression system. mRNAs were prepared from these vectors using T7 Megascript Kits* (Life Technologies, Inc.). |
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Product References:
Research performed using the Thermo Scientific Pierce Human In Vitro Protein Expression System.
- 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.
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