Protocol for in vitro protein expression using mRNA templates
Use the 1-Step Human Coupled DNA IVT kit to efficiently translate mRNA templates.
Atul Deshpande, Ph.D.;
Krishna Vattem, Ph.D.;
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.
Protocol for Protein Expression using mRNA Template
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.
- Thaw on ice the HeLa Lysate, Accessory Proteins, Reaction Mix [components of the 1-Step Human IVT Kit (Part No. 88881)] and mRNA. To hasten the thawing process, warm vials in gloved hands until half-way thawed, then place back on ice.
- Prepare reaction at room temperature in the proportions specified in Table 1. Add the reagents in the order listed into a 1.5mL nuclease-free tube. Gently mix the reaction after each reagent addition. To scale up the total reaction volume, maintain the specified proportions.
- Incubate the reaction for up to 5 hours at 30°C.
- Centrifuge reaction tube at 10,000 × g for 5 minutes to gather the contents.
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).
Protocol to Determine Level of Protein Expression from tGFP mRNA
Visualize or quantify expressed tGFP control protein using one of the following two methods:
- Quick visual detection: Place the GFP reaction tubes directly under a microscope or imaging equipment containing a FITC filter (ex/em: 482/512nm). Alternatively, spot a small volume (1-2μL) onto a piece of plastic wrap or laboratory film and visualize with fluorescent imaging equipment.
- Fluorescent plate reader: Pipette a small volume directly into a white or black 96- or 384-well plate. Evaluate signal using a fluorescent plate reader at ex/em: 482/512nm. To quantitate tGFP, compare the fluorescence to a standard curve of recombinant tGFP (Part No. 88899). See METHODS section for a specific example.
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 (e.g., Products AB513 and AB514, which are available from Evrogen).
RESULTS and DISCUSSION:
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.
Figure 1. In vitro translation (IVT) of mRNA template. Three independent pT7CFE-tGFP mRNA preparations (Lots 1, 2 and 3) were used to express tGFP protein. For each reaction, 2.25µg of mRNA was added to a 25µL reaction as described in the protocol above and incubated for 5 hours at 30°C. The amounts of tGFP expressed in the three lots were quantified by comparison to a standard curve of recombinant tGFP (Part No. 88899) using a plate-based assay (see METHODS). Error bars are for triplicate assay wells.
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.
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