The TnT® Coupled Reticulocyte Lysate Systems allow coupled in vitro transcription and translation of protein coding regions cloned downstream of the T7, SP6 or T3 RNA polymerase promoter in a eukaryotic environment. Researchers have a choice between several types of TnT® systems: the TnT® Coupled Reticulocyte Lysate Systems, TnT® Quick Coupled Transcription/Translation Systems, TnT® T7 Quick for PCR DNA and the Gold TnT® Express 96 Systems. These systems have been designed for different applications. To maximize protein yield, the rabbit reticulocyte lysate in each of these systems is supplemented with the components needed for efficient transcription and translation, such as magnesium and potassium ions, NTPs and calf liver tRNA, and is tested for efficient transcription and translation. In most cases, it is not necessary to add additional reagents to a TnT® reaction to achieve sufficient amounts of protein for downstream applications. However, in some cases, insufficient amounts of protein may be synthesized. We discuss the many parameters that can be optimized to improve yield of difficult templates.
A Complete Offering of in vitro TnT® Reticulocyte Lysate Systems
Promega offers many iterations of the TnT® Reticulocyte Lysate Systems, each of which have been optimized for different applications or DNA templates. The original TnT® Coupled Reticulocyte Lysate Systems (Cat.# L4950, L4600 and L4610) and the TnT® Quick Coupled Transcription/Translation Systems (Cat.# L1170 and L2080) transcribe and translate proteins from a plasmid DNA template in a single-tube format. Reaction volumes are typically 50µl but may be scaled up or down, depending upon the downstream application requirements. The TnT® Coupled Rabbit Reticulocyte Lysate System has 7 components, while in the TnT® Quick Coupled Transcription/Translation Systems, many of these components have been combined into one master mix. Therefore, less time and fewer pipetting steps are required to set up the TnT® Quick reactions. TnT® T7 Quick for PCR DNA (Cat.# L5540) is designed for the transcription and translation of linear, PCR-generated DNA templates, which often require higher potassium and magnesium concentrations than supercoiled templates. The yield from each of these systems can vary greatly, depending upon the nature of the DNA template and the protein product.
DNA Template Characteristics
The most influential factors for a successful TnT® reaction are related to the DNA template and include the context of the ATG translational initiation codon, sequence and structure of the 5´ untranslated region, the presence of a synthetic poly(A) tail, form (i.e., linear v. circular), amount of DNA template and template purity.
Long stretches of 5´ untranslated region (UTR) between the RNA polymerase promoter and the start codon can often have a negative impact on protein yield, especially if the 5´ UTR contains regions of strong secondary structure(1). The ideal template for in vitro transcription and translation will have a relatively short 5´ UTR (15–100bp) with no strong secondary structure. Also, the ideal template will have sequences that will produce a homopolymeric stretch of at least 30 A residues at the 3´ end of the RNA, which acts as a synthetic poly(A) tail when transcribed. Although a poly(A) tail is not required for translation, its presence can result in a two- to fivefold increase in protein synthesis levels(2). The TnT® Control DNAs supplied with the TnT® Systems encode a 30-nucleotide poly(A) tail.
RNA templates produced in a TnT® reaction will ideally have a Kozak consensus sequence (A/GNNAUGG, the translation start codon is in bold) at the site of translational initiation(3). However, a Kozak consensus sequence is not required for translation(2). The TnT® Control DNAs, which do not encode a Kozak consensus sequence, will typically produce 100–300ng of firefly luciferase protein per 50µl reaction. Additional AUG sequences present upstream of the true initiation codon can cause premature initiation, leading to shifts in the reading frame or larger-than-expected protein products, even if the AUG is not in the context of a Kozak consensus sequence. These additional AUG sequences may need to be deleted or mutated if translation is initiating at these sites.
Another important characteristic is the form of the DNA template. For T7 and T3 RNA polymerases, use of a circular DNA template does not produce significantly more protein than linear DNA for most templates. For SP6 RNA polymerase, the use of a circular DNA is much preferred over linear DNA and results in higher protein yields. For these reasons, we recommend using the circular form of most DNA templates. In cases where the DNA template does not have a transcription termination sequence, the DNA template can be linearized to generate an RNA of a specific size, but this is not usually necessary. Even in the absence of a transcription termination signal, the transcript has defined translation start and stop sites, and the size of the protein product is not affected. For linear DNA templates, we recommend using the T7- or T3-based TnT® Systems, not the SP6-based TnT® Systems. If linear DNA must be used, we recommend including at least 9–10bp upstream of a T7 or T3 RNA polymerase promoter(4) and at least 20bp upstream of the SP6 RNA polymerase promoter for efficient promoter binding(5).
The purity of the DNA template must be considered when the goal is maximal protein synthesis. Many plasmid DNA purification protocols involve the use of RNases. To inhibit any contaminating RNase activity in the DNA preparation, we recommend adding approximately 50 units of RNasin® Ribonuclease Inhibitor to a TnT® Coupled Reticulocyte Lysate reaction to avoid degradation of the in vitro transcripts and a reduction in protein yields. The master mixes supplied with the TnT® Quick Coupled Transcription/Translation Systems, Gold TnT® Express 96 Systems and the TnT® T7 Quick for PCR DNA System already contain RNasin® Ribonuclease Inhibitor. The DNA template should be free of known inhibitors, such as SDS, ethanol and guanidine. When a PCR product is used as a template in the TnT® T7 Quick for PCR DNA System, clean up is not required to remove the components of the amplification reaction(2). The amount of DNA template is not critical, but increasing volumes of DNA template can adversely affect protein yield if inhibitory contaminants are present in the template preparation. The standard TnT® protocols recommend 1µg of plasmid DNA or 2–5µl of an unpurified PCR product per 50µl reaction, depending upon the TnT® System. Satisfactory results have been obtained with 0.2–2µg of a suitable plasmid template or up to 7µl of a PCR product. The addition of more than the recommended amount of DNA template does not noticeably increase protein yield.
5´ Capped RNA is not Required
For in vitro translation kits, capping of the RNA is not usually required for protein synthesis(6)(7). However, for certain transcripts, capping of the RNA may be beneficial. The effect of capping on translation efficiency will be template-dependent. The TnT® Control DNA supplied with most of the TnT® Systems produces luciferase RNA, which does not need to be capped for translation. To determine if a 5´ cap is required for efficient translation of your RNA of interest, we recommend comparing the level of protein synthesis from capped and uncapped RNAs using the Rabbit Reticulocyte Lysate, Nuclease-Treated (Cat.# L4960).
In vitro translation of proteins greater than 100kDa in size can be difficult. Smaller-than-expected protein products may be seen when the TnT® reaction is analyzed on an SDS polyacrylamide gel. These smaller protein fragments are thought to be translational products of incomplete transcripts. Because more full-length transcript is often produced at lower temperatures, especially for GC-rich templates(8)(9), lowering the reaction temperature from the recommended 30°C to 22–25°C can increase the amount of full-length protein products. An alternative cause of smaller protein products is protease activity in the rabbit reticulocyte lysate(10). If reducing the reaction temperature does not improve the proportion of full-length protein, protease inhibitors can be added to a TnT® reaction, but the yield of protein may suffer, depending upon the protease inhibitor and the solvent. For example, experiments performed by Promega scientists have shown that 0.05M PMSF dissolved in ethanol and added to a TnT® reaction can reduce the percent incorporation of 35S methionine by three- to fourfold. This effect may be a direct inhibition by PMSF or inhibition by the ethanol solvent. Protease inhibitors that have been used successfully with the TnT® Systems include pepstatin, leupeptin(11)(12), chymostatin, antipain(11) and PefaBloc® SC(12), which is an aqueous alternative of PMSF. In vitro transcription and translation of proteins smaller than 15kDa can also be difficult. Small proteins can be degraded by a ubiquitin-dependent pathway within the rabbit reticulocyte lysate, and for this reason, we do not recommend the TnT® Systems for the synthesis of proteins smaller than 15kDa. Small proteins may be transcribed and translated more efficiently in the TnT® Coupled Wheat Germ Extract Systems.
Other problematic proteins include transmembrane or membrane-associated proteins, which may not be translated effectively in a TnT® reaction. Two approaches are often used to overcome this. Canine pancreatic microsomal membranes (Cat.# Y4041) or the detergent octaethylene glycol mono n-dodecyl ether (Nikkol) can be added to increase the yield of these proteins, presumably by minimizing the aggregation of these proteins(13). For most proteins, however, the addition of canine microsomal membranes or Nikkol detergent will reduce protein yield (see below).
The inhibitory effects of ethanol and other common DNA contaminants were discussed above. Glycerol is also inhibitory, so we include the appropriate phage RNA polymerase at a high concentration that has been optimized for each batch of rabbit reticulocyte lysate to minimize the amount of glycerol in the reaction. Supplementing the reaction with additional RNA polymerase, which is often stored in a buffer containing 50% glycerol, will lower protein yields. Other factors that can negatively affect the yield of a TnT® reaction include methanol, DMSO, formamide and a number of detergents, including Tween®-20, Nikkol, NP-40 and Triton® X-100(12). Oxidized glutathione, which can enhance disulfide bond formation and synthesis of active proteins, can also be detrimental at high concentrations(12). Other inhibitors include ammonium ions and metal ions. Heavy metal ions can inhibit protein synthesis by the activation of heme-regulated eIF-2 (HRI) kinase, which phosphorylates eIF-2α and shuts down initiation of protein synthesis(14)(15). Addition of increasing amounts of canine microsomal membranes to a TnT® reaction reduces the total number of polypeptides synthesized by approximately 50–80%. Even magnesium, which is necessary for transcription and translation, can inhibit at higher concentrations.
Potassium and Magnesium Concentrations
The salt concentration within an in vitro transcription/translation reaction can also have a dramatic effect on protein yield, depending upon the protein being expressed(16). Potassium and magnesium ions seem to have the greatest effect. For many plasmid templates, the optimal potassium concentration is approximately 125mM, and the optimal magnesium concentration is approximately 2.0mM(17). The TnT® Coupled Reticulocyte Lysate Systems provide potassium and magnesium concentrations near these values, and for most templates, there is little advantage of adding additional potassium and magnesium to the reaction. The potassium and magnesium concentrations in the TnT® Quick Coupled Reticulocyte Lysate Systems are somewhat lower than those in the TnT® Coupled Reticulocyte Lysate Systems. This results in higher yields of proteins from templates that require a lower salt concentration and provides more leeway when supplementing a TnT® reaction with potassium and magnesium to determine the optimal concentrations. For DNA templates that are expressing poorly even with favorable DNA template characteristics, we recommend supplementing a standard TnT® reaction with potassium (KCl) or magnesium (Mg acetate). This may increase protein yield. Assemble several reactions and increase the potassium concentration in 10–20mM increments and the magnesium concentration in 0.25mM increments. The optimal potassium and magnesium concentrations for in vitro transcription/translation from a PCR product is often higher, and so Promega developed the TnT® T7 Quick for PCR DNA specifically to meet these requirements of PCR-generated templates.
Effects of SDS Polyacrylamide Gel Electrophoresis Conditions
Prior to polyacrylamide gel electrophoresis, the newly synthesized protein must be denatured. Due to the high protein concentration in the rabbit reticulocyte lysate (100–150µg/µl), proteins are more prone to aggregation at higher denaturation temperatures. When a radioactive label, such as 35S methionine, is used, these aggregates can appear as a labeled high molecular weight band near the top of the gel. If this high molecular weight band is apparent and the protein of interest is not, dilute another 5µl aliquot of the TnT® reaction with 20µl of SDS sample buffer and denature the proteins at 60°C for 20 minutes, 70°C for 10 minutes or 80°C for 3–4 minutes instead of the recommended conditions of 100°C for 2 minutes. The milder denaturation conditions will reduce the risk of protein aggregation(18). Loading too much protein on an SDS-PAGE gel can also cause protein aggregation or cause the desired bands to be distorted. We recommend loading no more than 5µl of the TnT® reaction per lane.
Unexpected Bands Produced in a TnT® Reaction
When assembling a set of TnT® reactions, we recommend including a reaction with the empty vector. The presence of some vectors in the reaction can result in background bands(18), the size of which may vary with the vector. There are several background bands that can appear even in the absence of template DNA when an 35S methionine label is used in an in vitro transcription/translation reaction. These bands can complicate interpretation of results. The most prominent of these bands is a 25–30kDa band that is thought to be peptidyl tRNA, resulting from the translation of endogenous globin mRNA(17). This band can be minimized by treating the reaction with 50–100µg/ml of RNase A prior to the addition of SDS sample buffer and heat denaturation. Another background band that you may encounter has a molecular weight of approximately 42kDa; this product is thought to be the result of tRNA-dependent but ribosome-independent addition of labeled methionine to a pre-existing protein(17). The subunits of globin, which migrate as a 15–16kDa band, may appear near the bottom of the gel. Finally, larger-than-expected protein products can appear as a result of the SDS-PAGE conditions (see above).