Chapter 5: Protein Expression

Introduction

Cell-free systems for in vitro gene expression and protein synthesis have been described for many different prokaryotic (Zubay, 1973) and eukaryotic (Pelham and Jackson, 1976; Anderson et al. 1983) systems. Eukaryotic cell-free systems, such as rabbit reticulocyte lysate and wheat germ extract, are prepared from crude extract containing all the components required for translation of in vitro-transcribed RNA templates. Eukaryotic cell-free systems use isolated RNA synthesized in vivo or in vitro as a template for the translation reaction (e.g., Rabbit Reticulocyte Lysate Systems [Cat.# L4960, L4540] or Wheat Germ Extract Systems [Cat.# L4380]). Coupled eukaryotic cell-free systems combine a prokaryotic phage RNA polymerase with eukaryotic extracts and utilize an exogenous DNA or PCR-generated templates with a phage promoter for in vitro protein synthesis (TNT^® Coupled Reticulocyte Lysate [Cat.# L4600, L4610, L4950, L5010, L5020], TNT^® Quick Coupled Transcription/Translation Systems [Cat.# L1170, L2080], TNT^® T7 Quick for PCR DNA [Cat.# L5540] and TNT^® Wheat Germ Extract Systems [Cat.# L4120, L4130, L4140, L5030, L5040]).

Proteins translated using the TNT^® Coupled Systems can be used in many types of functional studies. TNT^® Coupled Transcription/Translation reactions have traditionally been used to confirm open reading frames, study protein mutations and make proteins in vitro for protein-DNA binding studies, protein activity assays, or protein-protein interaction studies. Recently, proteins expressed using the TNT^® Coupled Systems have also been used in assays to confirm yeast two-hybrid interactions, perform in vitro expression cloning (IVEC) and make protein substrates for enzyme activity or protein modification assays. For a listing of recent citations using the TNT^® Coupled Systems in various applications, please visit: www.promega.com/citations/

Transcription and translation are typically coupled in prokaryotic systems; that is, they contain an endogenous or phage RNA polymerase, which transcribes mRNA from an exogenous DNA template. This RNA is then used as a template for translation. The DNA template may be either a gene cloned into a plasmid vector (cDNA) or a PCR^(a)-generated template. A ribosome binding site (RBS) is required for templates translated in prokaryotic systems. During transcription, the 5´-end of the mRNA becomes available for ribosome binding and translation initiation, allowing transcription and translation to occur simultaneously. Prokaryotic systems are available that use DNA templates containing either prokaryotic promoters (such as lac or tac; E. coli S30 Extract System for Circular and Linear DNA [Cat.# L1020 and L1030] or a phage RNA polymerase promoter; E. coli T7 S30 Extract System for Circular DNA [Cat.# L1130]).

Most in vitro systems produce picomole (or nanogram) amounts of proteins per 50µl reaction. This yield is usually sufficient for most types of radioactive, fluorescent and antibody analyses, such as polyacrylamide gel separation, Western blotting, immunoprecipitation and/or, depending on the protein of interest, enzymatic or biological activity assays. For radioactive detection, a radioactive amino acid is added to the translation reaction and, after incorporation, the gene product is identified by autoradiography following SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Alternatively, non-radioactive labeling methods such as fluorescent, chemiluminescent or colorimetric detection may be used (i.e., Transcend™ and FluoroTect™ Systems; Sections XIII and XIV). If antibodies to the protein are available, then techniques such as immunoblotting or immunoprecipitation can be used. The functional activity of in vitro-translated products can often be detected directly in the reaction mixture. If protein purification is necessary, fusion of the protein to a purification “tag” allows the protein to be isolated from the in vitro translation reaction and subsequently studied.

Since protein synthesis reactions can be driven by RNA templates (translation; Section I.A) or DNA templates (coupled transcription/translation; Section I.B), the type of template is generally the first consideration when choosing an appropriate system. Promega translation and coupled transcription/translation systems are summarized in Tables 5.1 and 5.2, respectively. All systems provide reliable, convenient and efficient methods to initiate translation and produce full-size protein products.

Cell-free protein synthesis systems have become standard tools for the in vitro expression of proteins from cloned genes. Applications for in vitro expression systems include analysis of protein-protein and protein-nucleic acid interactions, mutational analysis, epitope mapping, in vitro evolutionary studies, protein truncation test (PTT) (Powell et al. 1993; Roest et al. 1993), clone verification, functional analysis, mutagenesis and domain mapping, ribosome display (Mattheakis et al. 1994; Hanes and Pluckthun, 1997) and in vitro expression cloning (IVEC) (Lustig et al. 1997; King et al. 1997), molecular diagnostics and high-throughput screening (Novac et al., 2004). In vitro expression systems also offer significant time savings over in vivo systems. The primary advantage of in vitro translation over in vivo protein expression is that in vitro systems allow the use of a defined template to direct protein synthesis. In vitro systems also have the ability to express toxic, proteolytically sensitive, or unstable gene products, and allow the specific labeling of gene products so that individual proteins can be monitored in complex reaction mixtures.

  mRNA Driven-Translation

The Rabbit Reticulocyte Lysate Translation Systems (Nuclease-treated and Untreated), Flexi^® Rabbit Reticulocyte Lysate System and Wheat Germ Extract System are all used for translation of mRNA. Table 5.1 summarizes these systems.

The Rabbit Reticulocyte Lysate, Nuclease-Treated, and the Flexi^® Rabbit Reticulocyte Lysate have been optimized for mRNA translation by adding several supplements. These include hemin, which prevents activation of the heme-regulated eIF-2a kinase (HRI); an energy-generating system consisting of pretested phosphocreatine kinase and phosphocreatine; and calf liver tRNAs, to balance the accepting tRNA populations, thus optimizing codon usage and expanding the range of mRNAs that can be translated efficiently. In addition both lysates are treated with micrococcal nuclease to eliminate endogenous mRNA, thus reducing background translation. The Flexi^® Rabbit Reticulocyte Lysate System provides greater flexibility of reaction conditions than the Rabbit Reticulocyte Lysate, Nuclease-Treated, by allowing translation reactions to be optimized for a wide range of parameters, including Mg²⁺ and K⁺ concentrations, and offers the choice of adding DTT. In contrast, the Rabbit Reticulocyte Lysate, Untreated, contains the cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, initiation, elongation and termination factors) but has not been treated with micrococcal nuclease. Untreated Rabbit Reticulocyte Lysate is used primarily for the isolation of translation components, as an abundant source of endogenous globin mRNA and to study protein synthesis of the endogenous globin mRNA. Untreated Rabbit Reticulocyte Lysate is not recommended for in vitro translation of specific mRNAs.

Wheat Germ Extract contains the cellular components necessary for protein synthesis (tRNA, ribosomes, initiation, elongation and termination factors). The extract is optimized further by the addition of an energy-generating system consisting of phosphocreatine and phosphocreatine kinase, spermidine to stimulate the efficiency of chain elongation and thus overcome premature termination, and magnesium acetate at a concentration recommended for the translation of most mRNA species. Only the addition of exogenous amino acids (including an appropriately labeled amino acid) and mRNA are necessary to stimulate translation. Finally, Potassium Acetate is supplied as a separate component so that the translational system may be optimized for a wide range of mRNAs.

DNA-Driven Coupled Transcription/Translation

Both eukaryotic and prokaryotic coupled transcription/translation systems are available from Promega. Table 5.2 summarizes these systems.

¹Only T7 linear templates.

²T7 promoter only, must use provided T7 TNT^® PCR Enhancer.

³Must be linearized for T7.

⁴SP6 and T3 promoters only.

The TNT^® Coupled Reticulocyte Lysate Transcription/Translation Systems and the TNT^® Quick Coupled Transcription/Translation Systems transcribe and translate proteins from plasmid templates using a single-tube format. The TNT^® Coupled Systems provide all the reaction components separately, including three separate amino acid mixtures: minus methionine, cysteine, or leucine. The TNT^® Quick Coupled System provides a master mix containing all the reaction components (including a minus methionine amino acid mix), thus saving time by requiring fewer pipetting steps. TNT^® T7 Quick for PCR DNA is specially formulated for transcription/translation of linear, PCR-generated templates, which often require higher potassium and magnesium concentrations than plasmid DNA. For transcription/translation of linear or PCR-generated templates with the TNT^® Quick Coupled System, a T7 TNT^® PCR Enhancer is provided and must be added to the reactions. Only linear templates containing the T7 promoter are recommended for the TNT^® Coupled Reticulocyte Lysate Transcription/Translation Systems. The Gold TNT^® Express 96 System is designed for high-throughput or IVEC applications and provides high-quality lysate predispensed into a 96-well plate. Transcription/translation requires only the addition to each well of a plasmid template containing the T7 or SP6 promoter, methionine (labeled or unlabeled) and Nuclease-Free Water.

The TNT^® Coupled Wheat Germ Extract System offers an alternative to the rabbit reticulocyte systems for eukaryotic coupled transcription/translation in a single-tube format. Unlike standard wheat germ extract translations, which commonly use RNA synthesized in vitro from SP6, T3 or T7 RNA polymerase promoters, the TNT^® Coupled Wheat Germ Extracts incorporate transcription directly in the translation mix.

The E. coli S30 Extract Systems simplify transcription/translation of DNA sequences cloned in plasmid or lambda vectors. The S30 extracts are prepared from E. coli B strains deficient in ompT endoproteinase and Ion protease activity. All three S30 systems contain an S30 Premix that includes all the components required for coupled transcription/translation except for amino acids. Separate amino acid mixtures minus methionine, cysteine or leucine are provided to facilitate radiolabeling of translation products. The E. coli S30 Extract Systems for Circular DNA and Linear DNA require that the gene of interest be under the control of a good E. coli promoter such as lambda PR, lambda PL, tac, trc or lacUV5. The E. coli T7 S30 Extract System for Circular DNA contains T7 RNA Polymerase as well as the components required for translation, thus simplifying transcription/translation of DNA sequences cloned into plasmid or lambda vectors containing a T7 promoter.

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Eukaryotic In Vitro Translation Systems

This section provides information on specific parameters you need to be aware of when using eukaryotic in vitro translation systems. We recommend reviewing the considerations that apply to the particular system being used before proceeding to the translation protocols detailed in Sections III–XII.

DNA Template Considerations

DNA purified using the PureYield™ Plasmid Midiprep System (Cat.# A2492, A2495) is sufficiently pure for use in TNT^® Rabbit Reticulocyte Lysate or Wheat Germ Extract reactions. A standard (50µl) TNT^® translation reaction requires 1µg of plasmid DNA as a template. However, 0.2–2.0µg of DNA template can provide satisfactory levels of translation, and adding more than 2µg of plasmid does not necessarily increase the amount of protein produced. For simultaneous expression from two or more DNA templates, we recommend adding approximately 0.5–1.0µg of each template, keeping the total amount of DNA added to 2µg or less.

Two template elements that are very helpful for increasing the efficiency of in vitro translation are an optimal Kozak sequence and a synthetic poly(A) tail of at least 30 nucleotides. Neither of these elements is required for translation using the TNT^® Systems, but each can help improve translation efficiency. The Kozak sequence (Kozak, 1986) serves to position the ribosome at the initiating AUG codon of the translated RNA. Poly(A)+ sequences have been reported to affect the stability and, therefore, the level of protein synthesized in Rabbit Reticulocyte Lysate (Jackson and Standart, 1990). We have noticed a two- to fivefold increase in luciferase production when the luciferase gene is cloned into the pSP64 Poly(A) Vector (Cat.# P1241). Another important consideration is the length of untranslated sequence between the transcription start site and the translation start site—a long 5´ untranslated region can form secondary structures, which may inhibit translation. In addition, there may be additional AUG sequences present in the untranslated region that could be recognized as a translation start site, resulting in fusion proteins or incorrect products. We recommend limiting the length of 5´ untranslated regions to less than 100bp.

Protein Labeling

Most researchers label in vitro translation products with [³⁵S]methionine. If the protein of interest contains only a few methionine residues, however, it may be necessary to label with an alternative radioactive amino acid or with a non-radioactive labeling system (Table 5.3). If there are sufficient cysteine or leucine residues in the protein, or if both methionine and cysteine or leucine will be used together to label the protein, then the appropriate amino acid mixture can be included in the TNT^® Coupled Reticulocyte Lysate Systems reaction. The TNT^® Coupled Systems contain amino acid mixtures lacking either methionine, cysteine or leucine. Amino Acid Mixture Minus Methionine and Cysteine is available separately (Cat.# L5511). Conversely, we don’t recommend using alternative radioactively labeled amino acids in the TNT^® Quick Coupled Systems, since the master mix contains all amino acids except methionine, and the labeling efficiency with other amino acids will be significantly reduced.

[³⁵S] Methionine

We recommend using a “translational grade” [³⁵S]methionine such as Amersham Biosciences Redivue™ (Amersham Biosciences Cat.# AG1094). We have obtained acceptable results using 1–4µl of [³⁵S]methionine (1,200Ci/mmol at 10mCi/ml). Depending upon the translational efficiency of the experimental RNA/DNA and number of methionines present in the protein, the amount of [³⁵S]methionine can be adjusted to balance exposure time against label cost. When using and storing [³⁵S]methionine, follow the manufacturer’s recommendations.

Non-Radioactive Protein Labeling

The Transcend™ Non-Radioactive Translation Detection Systems (Cat.# L5070 and L5080) and the FluoroTect™ Green_Lys in vitro Translation Labeling System (Cat.# L5001) can be used with any of the TNT^® Coupled or Quick Coupled Systems. These systems use a precharged lysine tRNA, which is incorporated into the translated protein. The Transcend™ System incorporates a biotinylated lysine, which can be detected by blotting and probing with streptavidin/AP or streptavidin HRP. The FluoroTect™ Reagent incorporates a fluorescently labeled lysine (BODIPY^®), which can be detected directly in the gel.

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TNT^® Quick Coupled Transcription/Translation Systems

Description

The TNT^® Quick Coupled Transcription/Translation Systems simplify the transcription/translation process by including all of the reaction components (RNA Polymerase, Nucleotides, salt and RNasin^® Ribonuclease Inhibitor) together with the reticulocyte lysate solution in a single TNT^® Quick Master Mix. The components of this Master Mix have been carefully adjusted to maximize both expression and fidelity for most gene constructs. When necessary, Magnesium Acetate and Potassium Chloride can be used to optimize in vitro translation reactions with the TNT^® Quick Systems. The inclusion of RNasin^® Ribonuclease Inhibitor directly in the Master Mix protects against potential disaster from the introduction of RNases carried over in the DNA solutions prepared using some miniprep protocols. The TNT^® Quick System is available in two configurations for transcription and translation of genes cloned downstream from either the T7 or SP6 RNA polymerase promoters. For a detailed protocol and background information on this system, please see Technical Manual #TM045.

Protocol

Materials Required:

• appropriate TNT^® Quick Coupled Transcription/Translation System (Cat.# L1170, L1171, L2080, or L2081)
• Nuclease-Free Water (Cat.# P1193)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non- radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)

To use these systems, 0.2–2.0µg of circular plasmid DNA containing a T7 or SP6 promoter, or a linearized plasmid or PCR-generated fragment containing a T7 promoter, is added to the TNT^® Quick Master Mix and incubated for 60–90 minutes at 30°C. The synthesized proteins are then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected (Figure 5.1).

The following is a general guideline for setting up transcription/translation reactions using plasmid or PCR-generated DNA as template. Examples of standard reaction setup using [³⁵S]methionine, Transcend™ Non-Radioactive Detection System or FluoroTect™ Green_Lys Systems are provided.

Plasmid DNA

Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube. After addition of all the components, gently mix by pipetting. If necessary, centrifuge briefly to return the reaction to the bottom of the tube. For the control reaction, use 1µl of the Luciferase Control DNA supplied.

Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

PCR-Generated DNA Templates

For PCR-generated templates, the T7 TNT^® PCR Enhancer should be included in the transcription/translation reaction.

Assemble the reaction components (below) in a 0.5ml or 1.5ml microcentrifuge tube. After addition of all the components, gently mix by pipetting. If necessary, centrifuge briefly to return the reaction to the bottom of the tube. For the control reaction, use 1µl of the Luciferase Control DNA supplied.

Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

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TNT^® Coupled Transcription/Translation Systems

Description

The TNT^® Coupled Reticulocyte Lysate Systems offer researchers an alternative for eukaryotic in vitro transcription and translation: a one-tube, coupled transcription/translation system. Standard Rabbit Reticulocyte Lysate or Wheat Germ Extract translations (Pelham and Jackson, 1976) commonly use RNA synthesized in vitro (Krieg and Melton, 1984) from SP6, T3 or T7 RNA polymerase promoters. The RNA is then used as a template for translation. The TNT^® Systems bypass many of these steps by incorporating transcription directly in the translation mix.

In most cases, the TNT^® System reactions produce significantly more protein (two- to sixfold) in a 1- to 2-hour reaction than standard in vitro Rabbit Reticulocyte Lysate or Wheat Germ Extract translations using RNA templates. In addition, TNT^® Lysates also can be used with microsomal membranes to study processing events (see Section XI).

Protocol

Materials Required:

• appropriate TNT^® System (Cat.# L4600, L4610, L4950, L5010, L5020, L4120, L4130, L4140, L5030 or L5040)
• Recombinant RNasin^® Ribonuclease Inhibitor (Cat.# N2511)
• radiolabeled amino acid
• Nuclease-Free Water (Cat.# P1193)

1. Assemble the reaction components (below) in a 0.5ml or 1.5ml microcentrifuge tube. After addition of all the components, gently mix by pipetting. If necessary, centrifuge briefly to return the reaction components to the bottom of the tube. For the control reaction, use 1µl of the Luciferase Control DNA supplied.

   Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

2. Incubate the reaction at 30°C for 90 minutes.

3. Analyze results.

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TNT^® Quick for PCR DNA—Coupled Transcription/Translation

Description

PCR-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. The TNT^® T7 Quick for PCR DNA System is optimized for expression of linear PCR products and requires no post-amplification purification of the template DNA. For a detailed protocol and background information about this system, please see Technical Manual #TM235.

PCR Primer Design

We have successfully used several software programs: OLIGO™ Primer Analysis software, PrimerSelect™ Expert Sequence Analysis software and Primer3 to assist in choosing primers for amplification.

5´ Primer

A T7 phage RNA polymerase promoter is required for transcription initiation from the PCR product DNA template. The T7 promoter may be either 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, the primer should be designed so that a Kozak consensus sequence is included in the PCR product. Typically, when amplifying from the 5´ UTR of a target cDNA, the native Kozak sequence is present (Kozak, 1987). However, when amplifying from an internal AUG, a Kozak consensus sequence must be added to the primer sequence. Recent literature suggests that there is polymorphism within the Kozak sequence and that certain sequences show increased translational efficiency in vitro and in vivo (Afshar-Khargan et al. 1999). Additional sequences (6–10 nucleotides) added upstream of the T7 consensus sequence ensure efficient RNA polymerase binding and RNA production.

3´ Primer

The 3´ primer typically matches the carboxy terminus of the gene or some position downstream from the translation termination codon and is generally 22–26 nucleotides in length. Some researchers have engineered an in-frame termination codon (e.g., TAA) into the 3´ primer sequence if the native termination codon is not present. The added termination codon may be useful for achieving multiple rounds of translation by allowing release of the ribosome from the peptidyl-tRNA. To allow release of the ribosome from the RNA template, we recommend designing the 3´ primer so that there are at least 20 nucleotides downstream of the translation termination codon. Beckler et al. (Beckler et al. 2000) discusses effective primer design.

Protocol

Materials Required:

• TNT^® T7 Quick for PCR DNA (Cat.# L5540)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non- radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)
• Nuclease-Free Water (Cat.# P1193)

To use TNT^® T7 Quick for PCR DNA, a PCR fragment containing a T7 promoter is added to the TNT^® T7 PCR Quick Master Mix and incubated for 60–90 minutes at 30°C. The synthesized proteins then can be analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography or phosphorimaging. Figure 5.1 shows a general guideline for setting up a transcription/translation reaction. Also provided below are examples of standard reactions using [³⁵S]methionine (radioactive), Transcend™ Non-Radioactive Detection Systems or the FluoroTect™ Green_Lys in vitro Translation Labeling System.

Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube. After addition of all components, gently mix by pipetting. If necessary, centrifuge briefly to return the reaction components to the bottom of the tube

Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

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TNT^® Coupled Wheat Germ Extract Systems—Coupled Transcription/Translation

Description

The TNT^® Wheat Germ Extract Systems are available in five configurations for transcription and translation of genes cloned downstream from the SP6, T3 or T7 RNA polymerase promoter. With these systems, a 50µl reaction is programmed with 0.2–2µg of template and incubated for 1.5 hours at 30°C. For a detailed protocol and background information about this system, please see Technical Bulletin #TB165. The following templates can be used with this system:

• Circular plasmid DNA containing a T3 or SP6 RNA polymerase promoter
• Linearized plasmid DNA containing a T7 RNA polymerase promoter
• Circular plasmid DNA containing both a T7 RNA polymerase promoter and T7 transcription terminator

Protocol

Materials Required:

• Appropriate TNT^® Coupled Wheat Germ Extract System (Cat.# L4120, L4130, L4140, L5030, L5040)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non- radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)
• Nuclease-Free Water (Cat.# P1193)
• RNasin^® Ribonuclease Inhibitor (Cat.# N2111 or N2511)

This section contains a protocol for coupled transcription/translation using the TNT^® Wheat Germ Extract. Multiple proteins can be expressed from different promoters in the same reaction by using multiple TNT^® RNA Polymerases. This allows greater flexibility in designing experiments for coexpression of multiple genes (DiDonato and Karin, 1993). T7/SP6 or T7/T3 polymerase may be added to the same reaction if they are from the same lot. In vitro-translated proteins expressed simultaneously in TNT^® Systems can be used to study protein-protein interactions. When using two DNA templates, add approximately 0.5–1.0µg of each template, keeping the total amount of DNA added to 2µg or less.

1. Assemble the reaction components in a 0.5ml or 1.5ml microcentrifuge tube. Gently mix the extract with a pipette tip upon addition of each component. If necessary, centrifuge briefly to collect the reaction at the bottom of the tube.

2. Incubate the reaction at 30°C for 60–120 minutes.

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Gold TNT^® Express 96 Transcription/Translation System

Description

The Gold TNT^® Express 96 Systems, available in either SP6 or T7 versions, are designed for transcription and translation of genes cloned downstream of either the SP6 or T7 RNA polymerase promoter. The systems consist of 96-well plates predispensed with 20µl of high-quality lysate per well. The systems' components are similar to the original TNT^® Coupled Transcription/Translation Systems; however, the Gold TNT^® System components meet a higher specification for expression of luciferase.

To use the Gold TNT^® Express 96 Systems, plasmid DNA containing the appropriate promoter is added, with either unlabeled or [³⁵S]-labeled methionine and Nuclease-Free Water (Cat.# P1193), to the wells of the plate. The proteins synthesized are then analyzed by functional testing or by SDS-polyacrylamide gel electrophoresis with subsequent detection by fluorescent imager, colorimetric/ chemiluminescent methods or autoradiography. For a detailed protocol and background information about this system, please see Technical Manual #TM054

Protocol

Materials Required:

• Gold TNT^® T7/SP6 Express 96 System (Cat.# L5600 or L5800)
• Nuclease-Free Water (Cat.# P1193)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non- radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)
• Additional substrates or factors as required (e.g., oligonucleotides, peptides, radiolabeled NTPs)
• Plate seals (e.g., Robbins Cat.# 1044-39-3)

The following is a general guideline for setting up a transcription/translation reaction using the Gold TNT^® Express 96 Transcription/Translation System.

1. For each reaction (i.e., one 20µl predispensed well), add the following components to yield a final reaction volume of 25µl.

2. Seal the plate containing the reactions with an adhesive plate seal.

3. Gently vortex the plate to mix.

4. Incubate the reaction at 30°C for 60–90 minutes.

5. Analyze the results of translation.

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Rabbit Reticulocyte Lysate Translation System, Nuclease-Treated

Description

The Rabbit Reticulocyte Lysate Translation System plays an important role in characterization of mRNA translation products, investigation of transcriptional and translational control, and co-translational processing of secreted proteins by the addition of microsomal membranes to the translation reaction. Rabbit Reticulocyte Lysate is prepared from New Zealand white rabbits injected with phenylhydrazine using a standard protocol to increase reticulocyte production (Pelham and Jackson, 1976). The reticulocytes are harvested, and any contaminating cells that could otherwise alter the translational properties of the final extract are removed. After lysis of the reticulocytes, the extract is treated with micrococcal nuclease to digest endogenous mRNA and thus reduce background translation to a minimum. The lysate contains the cellular components necessary for protein synthesis: tRNA, ribosomes, amino acids, and initiation, elongation and termination factors. Reticulocyte Lysate is further optimized for mRNA translation by adding several supplements as described in in Section I.A.

Rabbit reticulocyte lysate has been reported to contain a variety of post-translational processing activities, including acetylation, isoprenylation, proteolysis and some phosphorylation activity (Glass and Pollard, 1990). Processing events such as signal peptide cleavage and core glycosylation can be examined by adding canine microsomal membranes to a translation reaction (Andrews, 1987; Walter and Blobel, 1983; Thompson and Beckler, 1992)

The reaction conditions provided here are optimized for the Luciferase Control RNA supplied with the system and should be considered a starting point for experiments. However, many factors affect translation efficiency of specific RNAs in reticulocyte systems and should be considered when designing in vitro translation experiments. The optimal RNA concentration varies for different transcripts and should be determined empirically. In addition, the presence of certain nucleic acid sequence elements can have profound effects on initiation fidelity and translation efficiency; 3´-poly(A)+ sequences, 5´-caps, 5´-untranslated regions and the sequence context around the AUG start, or secondary AUGs in the sequence (Kozak, 1990). Lastly, optimal salt concentrations, particularly K⁺ and Mg²⁺ concentrations, may vary for different mRNAs and may need to be determined empirically (see Section IX).

RNA Template Considerations

Use a final concentration of 5–80µg/ml of in vitro transcripts produced with the RiboMAX™ Large Scale RNA Production Systems (Cat.# P1280 and P1300) for the translation. RNA from other standard transcription procedures may contain components at concentrations that inhibit translation. Therefore, a lower concentration, 5–20µg/ml of in vitro transcript, should be used with these systems. The optimal RNA concentration should be determined before performing experiments. Average preparations of mRNA stimulate translation about 10- to 20-fold over background (i.e., no exogenous RNA template). To determine the optimal concentration, serially dilute your RNA template first and then add the same volume of RNA to each reaction. This ensures that other variables are kept constant.

The presence of inhibitors can significantly reduce translation efficiency. Oxidized thiols, low concentrations of double-stranded RNA and polysaccharides are typical inhibitors of translation in rabbit reticulocyte lysate (Jackson and Hunt, 1983). To determine if inhibitors are present in your mRNA preparation, mix your RNA with Luciferase Control RNA and determine if translation of luciferase RNA is inhibited relative to a control translation containing only the luciferase RNA. Residual ethanol should also be removed from mRNA preparations and labeled amino acids before they are added to the translation reaction.

You may need to optimize the potassium and magnesium concentrations in your translation reactions. Addition of 0.5–2.5mM Mg²⁺ is generally sufficient for the majority of mRNAs. See Tables 5.4 and 5.5 for the concentrations of key components present in the lysate.

¹These amino acid concentrations should be used only as estimates. These values are not determined for individual lots of Rabbit Reticulocyte Lysate.

For many years, there have been discussions in the literature concerning which potassium salt is preferable in rabbit reticulocyte translation reactions. Potassium acetate (KOAc) often was used, rather than potassium chloride (KCl), because the chloride ion was shown to be inhibitory to translation, while higher levels of the acetate salt were not (Weber et al. 1977). However, several advantages of adding potassium chloride to rabbit reticulocyte translation reactions have been identified. Adding KCl, in addition to KOAc, can improve the fidelity of initiation from capped messages (Kozak, 1990). Uncapped in vitro-generated RNAs are reported to be translated with greater initiation fidelity using KCl instead of KOAc (Jackson, 1991). It also has been reported that high (120mM) levels of added KCl greatly enhance translational efficiency of EMCV (encephalomyocarditis virus) RNA (Jackson, 1991; Beckler, 1992). Although each RNA transcript will have its own optimal KCl concentration for translation, Table 5.6 can be used as a rough guideline (the volumes recommended are for a 50µl final reaction volume). If further optimization of salt concentrations is required, we recommend using the Flexi^® Rabbit Reticulocyte Lysate.

Protocol

Materials Required:

• Rabbit Reticulocyte Lysate, Nuclease-Treated (Cat.# L4960)
• RNasin^® Ribonuclease Inhibitor or RNasin^® Plus RNase Inhibitor (Cat.# N2111 or N2611)
• Nuclease-Free Water (Cat.# P1193)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non- radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)

The example below uses [³⁵S]methionine as the source of radiolabel; other isotopes may also be used (see Table 5.3). For the control reaction, use 2µl of the Luciferase Control RNA supplied. For a detailed protocol and background information about this system, please see Technical Manual #TM232.

1. Assemble the following reaction components in a 0.5ml or 1.5ml tube.

   Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

¹Final [³⁵S]methionine concentration = 0.8mCi/ml.

2. Incubate the reaction at 30°C for 60 minutes.

3. Analyze the results of translation.

   Note: The 70% concentration of lysate in the standard reaction is optimal for most applications. If required, the lysate can be diluted to 50% without a substantial reduction in translational efficiency. If optimal expression is desired in a 50% reaction, the levels of Mg²⁺ and K⁺ may need to be adjusted to account for the reduction in Mg²⁺ and K⁺ from the rabbit reticulocyte lysate.

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Flexi^® Rabbit Reticulocyte System—In Vitro Translation

Description

The Flexi^® Rabbit Reticulocyte Lysate System allows greater flexibility of reaction conditions than the standard Rabbit Reticulocyte Lysate System, Nuclease Treated. Different mRNAs commonly exhibit different optimum salt concentrations for translation. Furthermore, small variations in salt concentration can lead to dramatic differences in translation efficiency. The Flexi^® Rabbit Reticulocyte Lysate System allows optimization of a wide range of parameters, including Mg²⁺ and K⁺ concentrations, and offers the choice of adding DTT. To help optimize Mg²⁺ for a specific message, the endogenous Mg²⁺ concentration of each lysate batch is stated on the product insert. The Flexi^® System also offers the choice of three amino acid mixtures and includes a control RNA encoding the firefly luciferase gene. For a detailed protocol and background information about this system, please see Technical Bulletin #TB127.

Protocol

Materials Required:

• Flexi^® Rabbit Reticulocyte Lysate System (Cat.# L4540)
• RNasin^® Ribonuclease Inhibitor or RNasin^® Plus RNase Inhibitor (Cat.# N2111 or N2611)
• Nuclease-Free Water (Cat.# P1193)
• radiolabeled amino acid (for radioactive detection) or Transcend™ tRNA (Cat.# L5061) or Transcend™ Colorimetric (Cat.# L5070) or Chemiluminescent (Cat.# L5080) Translation Detection System (for non-radioactive detection) or FluoroTect™ Green_Lys in vitro Translation Labeling System (for fluorescent detection; Cat.# L5001)

The following is a general guideline for setting up a Flexi^® Lysate translation reaction. Also provided is an example of a standard reaction. The reaction uses [³⁵S]methionine as the radiolabel; other isotopes may also be used (see Table 5.3). For the positive control reaction, use 1–2µl of the Luciferase Control RNA supplied.

1. Assemble the following reaction components in a 0.5ml or 1.5ml tube.

   Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

2. Incubate the translation reaction at 30°C for 60–90 minutes.

3. Analyze the results of translation.

Optimization

The 66% concentration of lysate in the standard reaction is optimal for most applications. If desired, the lysate can be diluted 50 to 60% without a substantial reduction in translational efficiency. If optimal expression is desired in a reduced lysate concentration reaction, then the levels of Mg²⁺ and K⁺ must be adjusted to account for the reduction in Mg²⁺ and K⁺ from the rabbit reticulocyte lysate. The endogenous Mg²⁺ concentration of each lysate batch is listed on the product insert. Because the endogenous K⁺ concentration of each lysate batch is not determined, the optimal amount of K⁺ will have to be determined empirically.

Mg²⁺ is absolutely required and is the most critical component affecting translation. The range of Mg²⁺ for optimal translation is very narrow, and therefore, small changes in Mg²⁺ concentration can dramatically affect the efficiency of translation. Furthermore, each RNA transcript will exhibit an individual optimal Mg²⁺ concentration. To provide information useful for optimizing translation, the endogenous Mg²⁺ concentration of each lysate batch is stated on the product insert. For many RNA transcripts, this endogenous level should be very close to the optimal concentration. To determine if additional Mg²⁺ stimulates translation for a specific transcript, add 0–4µl of the provided Magnesium Acetate to a 0–2mM final added concentration in the standard 50µl reaction. High Mg²⁺ concentrations, though, can reduce the fidelity of translation and should be avoided (Snyder and Edwards, 1991).

No DTT is added to the Flexi^® Rabbit Reticulocyte Lysate during production. DTT can prevent the formation of disulfide bridges in proteins. If the alteration in structure affects the active site of the protein, the protein may be inactive. To study protein activity, we recommend that DTT not be added to the translation reaction. We have compared lysates prepared with or without added DTT. In those lysates prepared without DTT, we have added back DTT after thawing the stored lysate. We found no differences in translational efficiency or fidelity from these lysate combinations. If desired, 1µl of the provided 100mM DTT can be added to a 50µl (66%) lysate reaction to provide an identical concentration of DTT to that found in a standard Rabbit Reticulocyte Lysate reaction (2mM).

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Wheat Germ Extract—In Vitro Translation

Description

Wheat Germ Extract is prepared by grinding wheat germ in an extraction buffer, followed by centrifugation to remove cell debris. The supernatant is then separated by chromatography from endogenous amino acids and plant pigments that inhibit translation. The extract is treated with micrococcal nuclease to destroy endogenous mRNA and thus reduce background translation to a minimum. The extract contains the cellular components necessary for protein synthesis: tRNA, rRNA, and initiation, elongation and termination factors, and optimized further by the addition of several supplements as described in Section I.A.

Only the addition of exogenous amino acids (including an appropriate labeled amino acid) and mRNA are necessary to stimulate translation. Potassium acetate is supplied as an individual component so that the translational system may be additionally enhanced for a wide range of mRNAs.

The reaction conditions provided below are optimized for the BMV Control supplied with the system and should be considered a starting point for experiments. However, many factors affect translation efficiency of specific RNAs in Wheat Germ Extracts and should be considered when designing in vitro translation experiments (see Section II). For a detailed protocol and background information about this system, please see Technical Manual #TM230.

The optimal RNA concentration for translation should be determined before performing definitive experiments. To do this, serially dilute your RNA template first and then add the same volume of RNA to each reaction to ensure that other variables are kept constant.

Optimum potassium concentration varies from 50–200mM, depending on the mRNA. The optimal potassium concentration for translation of BMV RNA is 130mM. If this concentration results in poor translation of your sample mRNA, potassium levels should be adjusted to an optimum concentration. Certain mRNAs may also require altered magnesium concentration; optimum magnesium concentration for the majority of mRNAs is expected to fall in the range of 2–5mM. See Table 5.7 for the concentrations of key exogenous components of Wheat Germ Extract.

¹Additional potassium acetate may need to be added to optimize translation for each sample RNA.

Protocol

Materials Required:

• Wheat Germ Extract System (Cat.# L4380)
• RNasin^® Ribonuclease Inhibitor (Cat.# N2111 or N2511)
• radiolabeled amino acid
• Nuclease-Free Water (Cat.# P1193)

The reaction below uses [³⁵S]methionine; other isotopes may also be used (see Table 5.3). For the control reaction, use 2µl of BMV Control RNA.

1. Set up the following reaction in a 0.5 or 1.5 ml tube.

   Note: We recommend also including a negative control reaction containing no added template to allow measurement of background incorporation of labeled amino acids.

¹Final [³⁵S]methionine concentration = 0.5mCi/ml.

2. Incubate at 25°C for 60–120 minutes.

3. Analyze results.