by Atul Deshpande, Ph.D.; Krishna Vattem, Ph.D. - 05/22/13
The Thermo Scientific 1-Step Human Coupled IVT Kit (Part No. 88881) is a mammalian in vitro translation (IVT) system based on HeLa cell lysates for use with DNA templates. The kit contains all of the cellular components required for protein synthesis, including ribosomes, initiation factors, elongation factors and tRNA. Transcription and translation occur in a single reaction to produce protein from a supplied DNA template that is cloned into the pT7CFE1-based vector. The kit-system also can be used to efficiently sustain protein expression by translation from supplied mRNA templates for up to 5 hours.
The benefits of in vitro protein expression over traditional in vivo systems include the ability to express toxic or insoluble proteins, faster protein synthesis and protein labeling with modified amino acids. Using a vector containing the EMCV IRES element is critical for obtaining high expression levels in this human in vitro protein expression system. A gene of interest can be cloned into any pT7CFE1-based vector before transcribing it to make mRNAs and then translating it using components of the IVT kit. The optimized pT7CFE1 vectors contain a T7 RNA Polymerase promoter and an EMCV internal ribosome entry site (IRES) that promote high levels of in vitro protein expression that is cap-independent.
We developed a simple protocol to use components of the DNA-based 1-Step Human Coupled IVT Kit to express protein from mRNA templates. To assist in testing this procedure, we used a commercial in vitro transcription kit to produce a supply of “turbo-type” green fluorescent protein (tGFP) as a control mRNA (forthcoming product Thermo Scientific Pierce tGFP mRNA, Product 88880; contact us for information). Fluorescence of protein expressed from this tGFP mRNA template facilitates measurement and evaluation of the efficiency of the 1-Step Human Coupled IVT Kit to translate mRNA.
In this brief article, we present protocols for the translation and subsequent testing of mRNA, using tGFP mRNA as the template. Example data follow in the RESULTS and DISCUSSION section.
Take precautions to maintain an RNAse-free environment when working with mRNA. Thaw reagents immediately before use and keep on ice. Assemble the IVT reaction at room temperature. Promptly store any unused HeLa Lysate and other kit components at -80°C. Have ready previously prepared mRNA (e.g., tGFP mRNA), generated from an appropriate vector (see INTRODUCTION), and dilute to a concentration equal to 0.75µg/µL.
Following the translation reaction, keep the tube on ice for same-day analysis. Otherwise, store completed reactions at ≤ -20°C.
Table 1. Formulation of the IVT reaction. Except for the mRNA (template), components are from the Thermo Scientific 1-Step Human IVT Kit (Part No. 88881).
Visualize or quantify expressed tGFP control protein using one of the following two methods:
Be aware that tGFP control protein is from the copepod Pontellina plumata. This variety of GFP is not reactive to antibodies generated against Aequorea victoria GFP (i.e., EGFP or other EGFP mutants). However, it can be detected using anti- TurboGFP antibodies.
The described method (protocol) and test results (Figure 1) with tGFP mRNA both (1) demonstrate a general protocol for use with any mRNA template that contains the appropriate control elements of a pT7CFE Vector and (2) suggest a control strategy for testing new experimental systems.
To test, scale or modify this protocol with new mRNA templates, performing a parallel reaction with the tGFP mRNA (forthcoming Product 88880) enables easy validation of the reaction components and conditions.
Our example experiments demonstrate how mRNA templates that were transcribed from genes cloned into pT7CFE1 Vectors can be effectively translated using the Thermo Scientific 1-Step Coupled IVT Kit (Part No. 88881). We also describe how tGFP mRNA (forthcoming Product 88880) can be used as a control to test translation by this protocol.
mRNA production: tGFP DNA was cloned into pT7CFE1-CHis vector (Part No. 88860) and then transcribed using the Invitrogen™ MEGAscript™ In vitro Transcription Kit (Life Technologies, Product AM1334). The resultant mRNA was purified using one of the following two methods: (1) phenol-chloroform extraction and isopropyl alcohol precipitation according to instructions provided in the MEGAscript Kit, or (2) a spin-cup, cartridge-based, bind-and-elute protocol involving the Ambion™ MegaClear™ Kit (Life Technologies, Product AM1908), except that the number of wash steps was increased from two to three throughout.
mRNA quantitation: Aliquots of the purified mRNA samples were diluted 1:10, and the absorbance values at 260nm (A260) were measured using a NanoDrop™ 2000 UV-Vis Spectrophotometer. The concentration of each mRNA sample was calculated based on the accepted value that one A260 unit corresponds to 40μg/mL of single-stranded RNA. The mRNA samples were then adjusted to 0.75µg/µL for use in the in vitro translation (IVT) protocol.
In vitro translation (IVT): The data presented here were generated using the protocol outlined above with components of the Thermo Scientific 1-Step Human Coupled IVT Kit (Part No. 88881).
Expressed protein quantitation: 10µL aliquots of each reaction were diluted 15-fold with phosphate-buffered saline (PBS) containing 0.1% BSA to yield 150µL test samples. A set of standards was prepared from recombinant tGFP protein of known concentration (Part No. 88899) by serial dilution (50µg/mL to 0.78µg/mL) in 0.1% BSA-PBS. Triplicates of test samples and standards were pipetted into wells of a black 96-well plate and the fluorescence values measured by a Tecan™ Safire™ Fluorescent Plate Reader at ex/em: 482/512nm. Concentrations of test samples were calculated by reference to the standard curve.
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 100 µg/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 100 µg/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.
Features of the 1-Step Human Coupled IVT Kit:
Learn more about Thermo Scientific 1-Step Human In Vitro Protein Expression Kits
For Research Use Only. Not for use in diagnostic procedures.