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An article in four parts with references characterizing the use of the TNT® T7 Quick for PCR DNA (Cat.# L5540). 1. Introduction |
The importance of matching a genetic sequence with a particular protein function is becoming increasingly important as the amount of genome sequence information increases and becomes readily available to researchers. Multiple approaches are usually required to determine the function of any particular gene. Many of these studies can be performed using protein biochemistry and in vitro expression methodologies, such as in vitro translation.
Using DNA in a coupled transcription/translation format allowed the development of many in vitro expression applications, including gene construct verification, determination of protein function, detection of molecular interactions, detection of post-translational modifications, detection of disease-causing mutations by protein truncation test (PTT) or in vitro synthesized protein assay (IVSP), in vitro expression cloning(a) (IVEC) and ribosome display systems for cell-free protein evolution (1).
PCR(b)-generated DNA has increasingly become the template of choice for TNT® coupled transcription/translation reactions due to the ease of generating and using PCR products directly versus cloning specific targets by conventional means into plasmid vectors that contain genetic expression elements. TNT® T7 Quick for PCR DNA* was optimized for the expression of linear, unpurified PCR products. In comparison, the standard TNT® T7 Quick System* is optimized for plasmid DNA expression.
A T7 phage RNA polymerase promoter is required for transcription initiation from the PCR product DNA template. The T7 promoter may either be amplified from the plasmid vector containing the gene of interest, or the T7 promoter can be designed into the PCR product by addition to the forward or 5´ amplification primer. To ensure efficient translation initiation, a Kozak consensus sequence should also be present. The reverse or 3´ primer typically matches the carboxy terminus of the gene of interest and includes a stop codon (TAA, TGA or TAG). Promega Notes, Issue 74, discusses effective primer design (166 kilobyte pdf file).
Earlier work using TNT® T7 Quick for PCR DNA demonstrated that the system does not require any post-amplification purification of the template DNA and can produce up to five times more protein than other commercially available kits (Technical Manual #TM235). This article further characterizes the use of TNT® T7 Quick for PCR DNA with respect to template attributes, template purity and compatibility with other procedures.
Template Size
The 8.7kb APC (adenomatous polyposis coli) locus (2,3) was amplified by RT-PCR from HeLa cell total RNA and cloned into the pSP64 Poly(A) Vector (Cat.# P1241). Restriction enzyme digestion and partial sequencing verified that the clone carried the APC cDNA.
To investigate the effect of PCR product size on expression in this system, increasingly larger portions of the APC cDNA were amplified (Figure 1) using a forward primer containing a T7 RNA polymerase promoter with an ATG start codon and a reverse primer containing a stop codon (Figure 3, primers A and D). The PCR products ranged in size from 377–8,786bp, which should express proteins between 10–280kDa (Table 1).
Table 1. Expected Protein Size for APC PCR Fragments.
APC PCR Fragment # |
PCR Product |
Expected Protein |
1 |
377 |
10 |
2 |
707 |
20 |
3 |
1,376 |
40 |
4 |
2,039 |
60 |
5 |
2,669 |
80 |
6 |
3,335 |
100 |
7 |
5,000 |
150 |
8 |
6,668 |
200 |
9 |
8,000 |
240 |
10 |
8,786 |
280 |
Each APC PCR product was translated in vitro using TNT® T7 Quick for PCR DNA (5µl unpurified PCR product per reaction). The results are shown in Figure 2. TNT® T7 Quick for PCR DNA is able to translate proteins between 10–150kDa (lanes 1–7), although other smaller bands are visible in lane 6 (100kDa) and lane 7 (150kDa). No obvious bands at 200, 240 or 280kDa were detectable in lanes 8–10, but optimization of the translation reaction might allow expression of such large proteins (such as optimizing magnesium and potassium levels, lowering translation temperature, adding a poly(A) tail or increasing lysate concentration).
ATG Start Codon Context and Presence of Poly(A) Tail
Translation initiation occurs at an ATG (AUG) start codon present in the RNA template. For optimal translation initiation, the ATG codon should be present in a Kozak consensus sequence. The presence of a poly(A) tail on the 3´ end of a mRNA template also appears to enhance translation (see reference 4 for a review on translation initiation). To investigate the importance of these features when expressing DNA templates in TNT® T7 Quick for PCR DNA, two different APC products were amplified (encoding the 20kDa or the 60kDa protein) using forward primers that contained either a perfect Kozak consensus sequence (Figure 3, primer A), the endogenous APC ATG start codon, which is not a perfect Kozak consensus sequence (primer B), or a minimal T7 promoter and ATG start codon (primer C). In addition, the 60kDa APC template amplification either used a reverse primer that ended in a stop codon (primer D) or a stop codon followed by a poly(A)30 tail, which introduces a poly(A)30 tail into the RNA transcript (primer E).
An equal number of template molecules (1.8 × 1011) were used in each transcription/translation reaction. The results are shown in Figure 4. The 20kDa APC product showed reduced expression when only a minimal T7 promoter plus ATG start codon were incorporated into the PCR product. Expression of the 60kDa product was reduced when the endogenous APC start codon was used instead of a perfect Kozak consensus sequence. This level of expression was further reduced when only a minimal T7 promoter and ATG start codon were used. The reduced level of expression exhibited when a perfect Kozak consensus sequence was absent could be compensated for by including a poly(A)30 tail into the template RNA. The presence of a Kozak consensus sequence produces optimal expression, but a nonperfect start codon context can be compensated for by the addition of a poly(A)30 tail into the PCR product template DNA.
Templates Containing Deoxyuridine
To eliminate contamination of PCR reactions with previously amplified PCR product, many researchers include dUTP in their amplification reactions, and then degrade amplified product using UNG* (uracil N-glycosidase; 5). This may be important for those laboratories using PCR product translation for protein truncation test (PTT) reactions.
To investigate whether PCR product containing deoxyuridine could serve as a template for in vitro translation using the TNT® T7 Quick for PCR DNA system, two APC PCR products were amplified using either a nucleotide mix with or without dUTP*. As seen in Figure 5, Panels A and B, the APC PCR products generated using the dNTP nucleotide mix expressed well in this system. However, the APC PCR product generated using the dUTP nucleotide mix showed reduced expression, regardless of whether the PCR product was purified or unpurified. The unpurified 40kDa APC product (Figure 5, Panel B) generated with dUTP did express better than the 20kDa APC dUTP-containing product (Figure 5, Panel A) but was still much less than the unpurified 40kDa APC PCR product generated with a regular dNTP mix (Figure 5, Panel B). Similar results were obtained for the 80kDa APC PCR product (data not shown). The incorporation of dUTP into a PCR product appears to significantly inhibit expression in the TNT® T7 Quick for PCR DNA system.
The most likely block in the coupled transcription/translation reaction is the in vitro transcription step. To investigate whether T7 RNA polymerase fails to efficiently use a DNA template generated with dUTP, the purified 20kDa and 40kDa APC PCR products generated with either the regular dNTP mix or the mix containing dUTP were transcribed in vitro using the RiboMAX™ Large Scale RNA Production System for T7* (Cat.# P1300). These are the same PCR products that were translated in Figure 5. The expected size of in vitro transcript generated from the 20kDa APC PCR product #2 is 671 bases, and the expected in vitro transcript generated from the 40kDa APC PCR product #3 is 1,340 bases.
As shown in Figure 6, the APC PCR products generated with the regular dNTP mix produced a large amount of in vitro transcript, while the APC PCR products generated in the presence of dUTP produced relatively little in vitro transcript. The reduced protein expression using uridine-containing template in TNT® T7 Quick for PCR DNA can be explained by poor transcription from uridine-containing DNA template by T7 RNA polymerase.
PCR Product Purification and In Vitro Translation
Aliquots (50µl) of each APC PCR were purified using the Wizard® PCR Preps DNA Purification System* (Cat.# A7170, A2180) and eluted in 50µl Nuclease-Free Water (Cat.# P1193). Assuming 96–99% yield (Technical Bulletin #TB118), the PCR product concentration should be approximately equal before and after purification. Aliquots of either unpurified or purified APC PCR products (5µl) were then expressed in vitro using TNT® T7 Quick for PCR DNA*. The amount of radioactivity in each band was quantitated by phosphorimaging. Figure 7 shows that, for the PCR products tested, purification is not necessary.
Ribo m7G Cap Analog
The inclusion or contamination of in vitro translation reactions with cap analog has been theorized to inhibit translation by removing eIF-4 (eukaryotic initiation factor 4) from the reaction. eIF-4 is necessary for translation initiation as it recognizes and binds to the 5´ cap of mRNA and acts as a binding site for other necessary initiation factors (6). The T7 Luciferase Control DNA* (Cat.# L4821) was linearized to mimic a linear PCR product. Linearized T7 Luciferase Control DNA (500ng) was transcribed and translated in the TNT® T7 Quick for PCR DNA system in the presence of 0, 8, 16, 80, 160, or 800µM Ribo m7G Cap Analog (Cat.# P1712). As shown in Figure 8, even 800µM CAP Analog did not appear to inhibit expression of luciferase protein. It is possible that higher levels of CAP Analog or lower amounts of template DNA will reduce translation.
PCR Additives (Betaine, Glycerol, DMSO)
Many amplification targets require special PCR additives for amplification success. These include reagents such as betaine, glycerol and DMSO. To investigate whether such PCR additives would inhibit expression using TNT® T7 Quick for PCR DNA, linearized Luciferase T7 Control DNA was transcribed and translated in the presence of increasing amounts of betaine, glycerol or DMSO. The results shown in Figure 9 demonstrate that significant inhibition of luciferase protein expression is only seen at 2% DMSO, and some inhibition is seen with 1.6% glycerol. Betaine did not effect expression, even at a 100mM final concentration. Thus the TNT® T7 Quick for PCR DNA system seems robust in terms of carryover of PCR additives into the transcription/translation reaction.
Transcend™ Non-Radioactive Translation Detection System
To verify this translation system is compatible with non-radioactive detection, linearized Luciferase T7 Control DNA* was titrated into the TNT® T7 Quick for PCR DNA system in the presence of Transcend™ Biotinylated Lysine tRNA (Cat.# L5061). The results of this experiment, depicted in Figure 10 demonstrate that both colorimetric and chemiluminescent non-radioactive detection using Transcend™ tRNA can detect protein produced from as little as 10ng luciferase template DNA. As luciferase contains numerous lysine residues, proteins with fewer lysine residues will possibly exhibit less sensitive detection when using these non-radioactive detection methods.
Canine Pancreatic Microsomal Membranes (CMMs)
The compatibility of Canine Pancreatic Microsomal Membranes* (Cat.# Y4041) with the TNT® T7 Quick for PCR DNA system was investigated using the b-lactamase and a-mating factor Control RNAs, which are included with the CMMs (Cat.# Y4061, Y4071). Both RNAs were expressed in this system and processed as expected (Figure 11). The 31kDa b-lactamase protein undergoes signal peptide cleavage to 28kDa, while the 18kDa a-mating factor protein is N-glycosylated to 30kDa.
Figures for Characterization of TNT® T7 Quick for PCR DNA
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Figure 1. Agarose gel of PCR products amplified from an 8.7kb cDNA template. DNA fragment sizes are listed in Table 1. Lane M, 1kb DNA Ladder (Cat.# G5711). |
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Figure 2. In vitro translation of APC PCR products using TNT® T7 Quick for PCR DNA. The in vitro coupled transcription/translation reactions were performed as described in Technical Manual #TM235. Reactions were performed in the presence of 4µl [35S]methionine, and 2µl of each reaction were resolved per lane. All translation reactions were resolved on 4–12% Novex NuPAGE™ Tris-Bis gels in MES running buffer (Invitrogen). Negative control (–), no DNA template. Positive control (+), 100ng Luciferase T7 Control DNA* (Cat.# L4821). |
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Figure 3. Forward and reverse primers used for amplification of the APC PCR products. |
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Figure 4. Effect of the ATG start codon context and presence of poly(A) tail on expression of APC PCR products with TNT® T7 Quick for PCR DNA. The PCR products used as templates for coupled transcription/ translation reactions varied by the size of the protein produced (20kDa or 60kDa); the Kozak context of the start codon (perfect Kozak sequence [++], imperfect Kozak sequence [+] or no Kozak sequence [–]); and presence (+) or absence (–) of a poly(A) tail. An equal number of template molecules (1.8 × 1011) were used in each reaction. |
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Figure 5. Expression of 20kDa (Panel A) and 40kDa (Panel B) APC PCR products generated with or without dUTP in PCR. APC PCR products #2 (Panel A) and #3 (Panel B) were amplified using either a regular dNTP mix (200µM each dNTP) or a mix of nucleotides containing dUTP (200µM each dATP, dCTP, and dGTP and 600µM dUTP). Aliquots of each PCR were purified using the Wizard® PCR Preps DNA Purification System* (Cat.# A2180) and then quantitated using absorbance at 260nm. The PCR products were then translated in vitro using either 5µl unpurified product, 5µl purified product or a constant amount of DNA template for each product (600ng). |
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Figure 6. In vitro transcription using the RiboMAX™ System (Cat.# P1300) of APC PCR products generated with or without dUTP in PCR. An aliquot (1µg) of each PCR product was transcribed in vitro in a reaction volume of 25µl as described in Technical Bulletin #TB166. Following transcription, the reaction volume was increased to 50µl by the addition of 25µl nuclease-free water, and treated with 1µl RQ1 RNase-free DNase for 15 minutes at 37°C, then extracted once with phenol/chloroform/isoamyl alcohol and the supernatant passed over a micro G50 column (Amersham) to remove unincorporated rNTPs as well as degraded template DNA. An aliquot of each transcription reaction (12µl) was then mixed with 8µl RNA loading dye (Ambion) and analyzed on a 1.4% agarose/1X TAE gel. The RNA samples were denatured for 10 minutes at 65°C prior to loading onto the gel. |
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Figure 7. Effect of PCR product purification on expression in TNT® T7 Quick for PCR DNA. PCR products were used directly in the expression reaction or were purified using the Wizard® PCR Preps DNA Purification System (Cat.# A2180). The templates used were as described in Figure 4. |
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Figure 8. Effect of Ribo m7G Cap Analog on the expression of luciferase protein in TNT® T7 Quick for PCR DNA. The T7 luciferase control DNA was linearized using Eco47 III, which cleaves approximately 130bp downstream of the poly(A) tail. Linearized Control DNA (500ng) was transcribed and translated using TNT® T7 Quick for PCR DNA in the presence of the indicated amount of CAP Analog. |
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Figure 9. Effect of various PCR additives on luciferase expression using TNT® T7 Quick for PCR DNA. Linearized Luciferase T7 Control DNA was transcribed and translated using TNT® T7 Quick for PCR DNA in the presence of the indicated amounts of betaine, glycerol or DMSO. |
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Figure 10. Titration of linearized luciferase plasmid DNA into TNT® T7 Quick for PCR DNA using Transcend™ Biotinylated Lysine tRNA incorporation. Linearized Luciferase T7 Control DNA was titrated (0, 1, 10, 100 or 500ng) into the TNT® T7 Quick for PCR DNA system in the presence of Transcend™ Biotinylated Lysine tRNA (1.5µl per reaction). Equal amounts of each reaction were separated on 4–12% polyacrylamide gels, transferred to supported nitrocellulose membrane, and developed using the appropriate AP- or HRP-streptavidin conjugate and either a colorimetric (Panel A) or chemiluminescent (Panel B) detection reagent as described in Technical Bulletin #TB182. Chemiluminescent detection used a two-minute exposure. |
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Figure 11. Expression and processing of b-lactamase and a-mating factor RNAs in TNT® T7 Quick for PCR DNA in the presence or absence of CMMs. A 100ng aliquot of each Control RNA (Cat.# Y4061, Y4071) was translated in the presence or absence of 2µl of CMMs in a 25µl TNT® T7 Quick for PCR DNA reaction. Each reaction (4µl) was then resolved on a 4–12% polyacrylamide gel, dried and exposed to film for 1.25 hours. |
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