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Protein Expression

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Chapter Sections

Introduction

Eukaryotic Cell-Free Translation Systems

Eukaryotic Cell-Free Expression Systems

Rabbit Reticulocyte Lysate Translation Systems

Wheat Germ Extract—Cell-Free Translation

Co-Translational Processing Using Canine Pancreatic Microsomal Membranes

Prokaryotic Cell-Free Translation Systems

Transcend™ Non-Radioactive Translation Detection System

FluoroTect™ Green_Lys in vitro Translation Labeling System

References

All Chapters

Nucleic Acid Amplification

RNA Interference

Apoptosis

Cell Viability

Protein Expression

Expression Analysis

Cell Signaling

Bioluminescence Reporters

DNA Purification

Cell Imaging

Protein Purification and Analysis

Transfection

Cloning

DNA-Based Human Identification

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 (Figure 5.1) ( 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 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 VIII and IX, respectively). 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. For assistance in choosing a cell-free protein expression system, the Cell-Free Protein Expression Product Selector is available.

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 (/multimedia/tnt01.htm) and protein:nucleic acid interactions (/multimedia/tnt02.htm). In addition, these systems can be used for 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 Cell-Free Expression

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

¹Expected yield for 50µl reaction volumes.

²Only T7 linear templates.

³T7 promoter only, must use provided T7 TNT^® PCR Enhancer.

⁴Must be linearized for T7.

⁵SP6 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 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 Cell-Free 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–VII.

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 PerkinElmer EasyTag™ L-[³⁵S]methionine (PerkinElmer Cat.# NEG709A). 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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Eukaryotic Cell-Free Expression Systems

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 VII).

TNT^® SP6 High-Yield Protein Expression System

The TNT^® SP6 High-Yield Protein Expression System uses a high-activity wheat germ extract supplemented with SP6 RNA polymerase and other components to express 10–100µg/ml of soluble protein in a one-tube coupled transcription/translation format. The system produces substantially more protein than conventional wheat germ systems while maintaining the ease of use that cell-free and coupled transcription and translation conditions afford. Following a two-hour incubation, the expressed protein can be used directly in downstream applications; no protein purification steps are necessary. For a detailed protocol and background information on this system, please see Technical Manual #TM282.

TNT^® T7 Insect Cell Extract Protein Expression System

The TNT^® T7 Insect Cell Extract Protein Expression System is a single-tube, coupled transcription/translation system for the cell-free expression of proteins. The extract is made from the commonly used Spodoptera frugiperda Sf21 cell line (Ezure et al. 2006) However, the extract contains no endogenous glycosylation machinery. All necessary components are present in the TNT^® T7 ICE Master Mix (ICE = insect cell extract). Protein is expressed from genes cloned downstream of the T7 polymerase promoter. The best expression will be achieved using vectors that contain the baculovirus polyhedrin 5´ and 3´ untranslated region (Suzuki et al. 2006). Two vectors, the pF25A and pF25K ICE T7 Flexi^® Vectors (Cat.# L1061 and L1081), have been designed specifically to produce optimal protein yields in this system. For a detailed protocol and background information on this system, please see Technical Manual #TM305.

The pF25A and pF25K ICE T7 Flexi^® Vectors are compatible with the Flexi^® Cloning System (Cat.# C8640), which allows easy transfer of sequences to and from additional Flexi^® Vectors. Additional Flexi^® Vectors are available for use with other protein expression systems. For a detailed protocol and background information on this system, please see Technical Manual #TM254.

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

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.

TNT^® Quick Coupled Transcription/Translation Systems

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

• 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 5.2).

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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Rabbit Reticulocyte Lysate Translation Systems

Standard Rabbit Reticulocyte Lysate Translation

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 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)

Flexi^® Rabbit Reticulocyte System—In Vitro Translation

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.

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. 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).

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.

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—Cell-Free 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.

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Co-Translational Processing Using Canine Pancreatic Microsomal Membranes

Description

Microsomal vesicles are used to study co-translational and initial post-translational processing of proteins (Rando, 1996; Han and Martinage, 1992; Chow et al. 1992). Processing events such as signal peptide cleavage, (MacDonald et al. 1988), membrane insertion (Ray et al. 1995), translocation and core glycosylation (Bocco et al. 1988) can be examined by translation of the appropriate mRNA in vitro in the presence of microsomal membranes. Processing and glycosylation events may also be studied by transcription/translation of the appropriate DNA in TNT^® Rabbit Reticulocyte Lysate Systems when used with microsomal membranes. To assure consistent performance with minimal translational inhibition and background, Promega microsome preparations have been isolated free from contaminating membrane fractions and stripped of endogenous membrane-bound ribosomes and mRNA. Membrane preparations are assayed for both signal peptidase and core glycosylation activities using two different control mRNAs. The two control mRNAs supplied with the Canine Microsomal Membranes are the precursor for β-lactamase (or ampicillin resistance gene product) from E. coli and the precursor for α-mating factor (or α-factor gene product) from S. cerevisiae.

For a detailed protocol and background information about this system, please see Technical Manual #TM231.

General Protocols for Translation with Microsomal Membranes

While the reaction conditions described here are suitable for most applications, the efficiency of processing using other membranes may vary. Thus, reaction parameters may have to be altered to suit individual requirements. In general, increasing the amount of membranes in the reaction increases the proportion of polypeptides translocated into vesicles but reduces the total amount of polypeptide synthesized. The amount of protein produced in lysates using Microsomal Membranes will be less than the amount produced in lysate alone. Depending on the DNA or RNA used, translation efficiency can be expected to drop between 10–50% in the presence of Microsomal Membranes.

Materials Required:

• Canine Pancreatic Microsomal Membranes (Cat.# Y4041)
• appropriate Reticulocyte Lysate System
• Recombinant RNasin^® Ribonuclease Inhibitor (Cat.# N2511)
• radiolabeled amino acid
• Nuclease-Free Water (Cat.# P1193)

1. Mix the following components on ice, in the order given, in a sterile 1.5ml microcentrifuge tube.

¹For the control reactions, use pre β-lactamase and α-factor mRNA at 0.1µg/ml.

2. Incubate for 90 minutes at 30°C.

3. Analyze results.

Analysis of Results

When using 1.8µl of Microsomal Membranes per 25µl of translation mix, 90% of pre-β-lactamase will be processed to β-lactamase. The same amount of membranes will process 75–90% of α-factor to core glycosylated forms of α-factor. Upon SDS-PAGE, the precursor for β-lactamase migrates at 31.5kDa and the processed β-lactamase at 28.9kDa. The precursor for the α-factor migrates at 18.6kDa, and the core-glycosylated α-factor has a molecular weight of 32.0kDa but will migrate faster than the β-lactamase precursor (Figure 5.4).

In some cases, it is difficult to determine if efficient processing or glycosylation has occurred by gel analysis alone. These alternative assays for detecting co-translational processing events may be useful. A general assay for co-translational processing uses the protection afforded the translocated protein domain by the lipid bilayer of the microsomal membrane. In this assay, protein domains are judged to be translocated if they are observed to be protected from exogenously added protease. To confirm that protection is due to the lipid bilayer, addition of 0.1% non-ionic detergent (such as Triton^® X-100 or Nikkol) solubilizes the membrane and restores susceptibility to protease. Many proteases have proven useful for monitoring translocation in this fashion including protease K and trypsin (final concentration 0.1mg/ml; Gross et al. 1988).

An alternative procedure uses endoglycosidase H to determine the extent of glycosylation of translation products (Andrews, 1987). In cell-free systems, N-linked glycosylation occurs only within intact microsomes. Endoglycosidase H cleaves the internal N-acetylglucosamine residues of high mannose carbohydrates, resulting in a shift in apparent molecular weight on SDS-polyacrylamide gels to a position very close to that of the nonglycosylated species. The reaction conditions (0.1% SDS, 0.1M sodium citrate [pH 5.5] incubation at 37°C for 12 hours) are not compatible with those required to maintain membrane integrity. For this reason, translocated polypeptides are not “protected” from digestion with endoglycosidase H.

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Prokaryotic Cell-Free Translation Systems

This section provides information on specific parameters that require optimization for prokaryotic in vitro translation reactions. We recommend that you review the considerations that apply to the particular system being used before proceeding to the translation protocols (Section VII.D).

Description

The S30 extracts in the E. coli S30 Extract Systems are prepared by modifications of the methods described by Zubay (Zubay, 1973; Zubay, 1980; Lesley et al. 1991). The E. coli S30 Extract System for Linear Templates (Cat.# L1030) is prepared from E. coli B strains deficient in ompT endoproteinase and lon protease activity. This results in greater stability of expressed proteins, which would otherwise be degraded by proteases if expressed in vivo (Pratt, 1984; Studier and Moffatt, 1986). The E. coli B strain used to produce the S30 Extract for Linear Templates is also deficient in exonuclease V (the recBCD enzyme). The recD mutation of this strain produces a more active S30 Extract System for Linear DNA than previously described nuclease-deficient recBC-derived S30 extracts (Lesley et al., 1991; Pratt, 1984; Chen and Zubay, 1983). However, the S30 Extract for Linear Templates is less active than the S30 Extract System for Circular DNA and T7 S30 Extract. The E coli S30 Extract System for Circular DNA also allows higher expression levels of proteins that are normally expressed at low levels in vivo due to the action of host-encoded repressors (Collins, 1979). For a detailed protocol and background information about the E coli S30 Extract Systems, please see Technical Bulletin #TB092, Technical Bulletin #TB102 and Technical Bulletin #TB219.

The E. coli T7 S30 Extract System for Circular DNA simplifies transcription/translation of DNA sequences cloned in plasmid or lambda vectors containing a T7 promoter by providing an extract that contains T7 RNA Polymerase for transcription and all necessary components for translation. The researcher only supplies the cloned DNA containing a T7 promoter and a ribosome binding site.

The E. coli S30 Systems contain an S30 Premix Without Amino Acids that is optimized for each lot of S30 Extract and contains all other required components, including rNTPs, tRNAs, an ATP-regenerating system, IPTG and appropriate salts. Amino acid mixtures lacking cysteine, methionine or leucine are provided to facilitate radiolabeling of translation products.

The most common application of E. coli S30 Extract Systems is the synthesis of small amounts of radiolabeled protein. The synthesis of a protein of the correct size is a useful way to verify gene products. Proteins expressed in the E. coli S30 Extract Systems may also be used for a variety of functional transcription and translation studies. Additional applications of the E. coli S30 Extract Systems include synthesis of small amounts of radiolabeled protein for use as a tracer in protein purification (Promega Notes 26, 1990) and incorporation of unnatural amino acids into proteins for structural studies (Noren et al. 1989).

  Template Considerations

Use only highly purified DNA templates (e.g., CsCl- or gel-purified) and avoid adding high concentrations of salts or glycerol with the DNA template. The activity of the E. coli S30 Systems may be inhibited by NaCl (≥50mM), glycerol (≥1%), and by small amounts of Mg²⁺ (1–2mM) or potassium salts (50mM). The DNA template should be ethanol-precipitated with sodium acetate rather than ammonium acetate. Protein yields from the E. coli S30 Extract Systems vary with the template and the conditions used. Typical protein yields range from 50–250ng per 50µl reaction.

S30 Extract and T7 S30 Extract for Circular DNA

Expression of cloned DNA fragments in the E. coli S30 Extract System for Circular DNA requires that the gene be under the control of a good E. coli promoter. Examples of such promoters include lambda PR, lambda PL, tac, trc and lacUV5. Expression of cloned DNA fragments in the T7 S30 Circular System requires that the gene be under the control of either a T7 or a good E. coli promoter. Expression levels from T7 promoters are typically higher than that from E. coli promoters in this extract. Expression from E. coli promoters can be inhibited by the addition of rifampicin to the extract; transcription by T7 RNA Polymerase is resistant to rifampicin.

S30 Extract for Linear DNA or RNA

Expression of gene products from linear DNA containing supercoiling-sensitive promoters can be reduced in the S30 System by up to 100-fold (Chen and Zubay, 1983). Examples of good promoters that are supercoiling-insensitive include lacUV5, tac, λ and λPR. DNA from other prokaryotic species may not contain promoters that direct transcription in the E. coli S30 Extract System for Linear Templates. RNA generated in vitro from cloned genes lacking an E. coli promoter is also suitable. Larger templates, such as bacteriophage lambda DNA, may also be used.

PCR-Generated Templates

PCR technology has introduced many methods for site-specific in vitro mutagenesis. Combining PCR with phage λ exonuclease treatments has produced mutated fragments larger than 2.5kb (Shyamala and Ames, 1991). PCR products can be added to the E. coli S30 Extract System for Linear Templates for rapid confirmation of expected protein size or activity.

Avoid contaminating the S30 Extract reaction with the wrong PCR product or primer dimers. If agarose gel analysis indicates that your PCR reaction produced a unique band, primer dimers can be removed by ethanol precipitation with sodium acetate. Otherwise, PCR-amplified DNA should be gel purified before use.

Restriction Enzyme-Digested Templates

For restriction enzyme-digested DNA, 10–20µg of DNA should be digested in a 100–200µl reaction volume. Ethanol precipitate and resuspend the DNA at a concentration of 1µg/µl in TE buffer or water. Add 2–4µg of this DNA directly to the S30 reaction. However, if the desired results are not obtained, the DNA should be further purified by phenol extraction followed by ethanol precipitation.

RNA Templates

The amount of in vitro RNA added to the extract can vary from 10–100µg. For synthesizing milligram quantities of highly pure, “translatable” RNA, we recommend using one of the RiboMAX™ Large Scale RNA Production Systems (Cat.# P1280 and P1300; RiboMAX™ Large Scale RNA Production Systems—SP6 and T7 Technical Bulletin, #TB166.

S30 T7 High-Yield Protein Expression System

The S30 T7 High-Yield Protein Expression System is an E. coli extract-based cell-free protein synthesis system. It simplifies the transcription and translation of DNA sequences cloned in plasmid or lambda vectors containing a T7 promoter by providing an extract that contains T7 RNA polymerase for transcription and all necessary components for translation. This system can produce high levels of recombinant proteins (up to hundreds of micrograms of recombinant protein per milliliter of reaction) within an hour using a vector containing the sequence of interest, a T7 promoter and a ribosome-binding site (RBS).

The S30 T7 High-Yield Protein Expression System contains the T7 S30 Extract,Circular, which is prepared by modifications of the method described by Zubay (Zubay, 1973 and Zubay, 1980) from an E. coli strain B deficient in OmpT endoproteinase and lon protease activity. This results in greater stability for translated proteins that would otherwise be degraded by proteases if expressed in vivo (Studier and Moffatt, 1986; Pratt, 1984). An optimized S30 Premix Plus provides all other required components, including amino acids, rNTPs, tRNAs, an ATP-regenerating system, IPTG and appropriate salts to express high levels of recombinant proteins.

Using this system, protein can be expressed in volumes as small as 5μl for high-throughput screening in a 96-well plate format. The amount of protein synthesized increases proportionally with reaction volume (up to 250μl). Because it is a cell-free system, the S30 T7 High-Yield Protein Expression System can be used to express proteins that are toxic to E. coli cells. Finally, small amounts of target protein can be purified using affinity tags (e.g., metal-affinity tag) for downstream analysis. For a detailed protocol and background information about the S30 T7 High-Yield Protein Expression System, please see Technical Manual #TM306.

  E. coli S30 Extract Systems Protocol

Materials Required:

• appropriate E. coli S30 Extract System (Cat.# L1020, L1030 or L1130)
• radiolabeled amino acid
• Nuclease-Free Water (Cat.# P1193)

The following protocol is designed for use with the E. coli S30 Extract Systems for Circular (including the T7 System) or Linear DNA Templates and [³⁵S]methionine as the radiolabel. Other radioisotopes may also be used (see Table 5.3). For positive control reactions, use 4µl of the Control DNA provided. For multiple reactions, create a master mix by combining the appropriate volumes of Amino Acid Mixture Minus Methionine (or Cysteine or Leucine), S30 Premix Without Amino Acids, radiolabeled amino acid (optional), S30 Extract and Nuclease-Free Water. Aliquot the master mix into 1.5ml microcentrifuge tubes and initiate the reactions by adding the DNA template to the tubes.

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

2. Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to collect the reaction mixture at the bottom of the tube.

3. Incubate the reaction at 37°C for 1–2 hours.

4. Stop the reaction by placing the tubes in an ice bath for 5 minutes.

5. Analyze the results of the reaction.

Optimization

The amount of DNA added should be optimized. In general, reactions should not contain more than 4µg of DNA. An increased amount of DNA can result in higher incorporation of label but can also increase the number of internal translational starts or prematurely arrested translation products detected. For the positive control reactions, use the PinPoint™ Control Vector DNA for T7 systems and pBESTluc™ DNA for non-T7 systems. Template DNA/RNA and water purity are extremely important. If efficiencies are low, examine the quality of the template DNA and water. See Section VII.B for general template considerations.

For the T7 S30 Extract, transcription by the endogenous E. coli RNA polymerase can be inhibited by the addition of the antibiotic rifampicin, while transcription by the phage T7 RNA Polymerase is unaffected. To inhibit the endogenous RNA polymerase, add 1µl of a 50µg/ml solution of rifampicin in water prior to the addition of the DNA template to the reaction. Addition of excess rifampicin is unnecessary and may decrease protein synthesis. The T7 S30 Extract contains nuclease activity, which prevents the use of linear DNA templates such as PCR products in the reaction. PCR products containing a T7 promoter and a ribosome binding site can be used by adding a small amount (1µl) of the T7 S30 Extract to the E. coli S30 Extract for Linear DNA.

Controls

The S30 Extracts for Linear and Circular DNA use pBESTluc™ DNA (Linear or Circular, respectively) as a control to synthesize luciferase. Luciferase protein migrates at 61kDa in denaturing gels. An apparent internal translation start results in a second major gene product of 48kDa. The control plasmid also contains the gene for ampicillin resistance (β-lactamase). β-lactamase may appear as a faint band migrating at 31.5kDa. Unlabeled luciferase is used in a luminescence assay to monitor the efficiency of the S30 reaction. To generate unlabeled luciferase, use a complete Amino Acid Mixture, rather than a Minus Amino Acid Mixture, and omit the radiolabeled amino acid.

The T7 S30 System for Circular DNA uses the PinPoint™ Control Vector DNA template as a positive control. Translation of this vector will result in the synthesis of the proteins shown in Figure 5.5. The largest molecular weight band corresponds to the PinPoint™-CAT fusion protein (39kDa). A prominent band corresponding to β-lactamase (28kDa) migrates below the PinPoint™-CAT fusion. Expression of β-lactamase is significantly higher in the T7 S30 Extract. This is the result of transcription from the T7 promoter upstream of the fusion protein, which reads through the ampicillin resistance gene. Some full-length CAT protein is also observed, which is probably due to an internal translation initiation site. For a negative control, omit the DNA from the reaction. Use the negative control to determine background radiolabel incorporation.

Reaction Temperature

The reaction may be incubated between 24–37°C. The fastest linear rate occurs at 37°C for approximately 2 hours, although the reaction will continue for several hours at a slower rate. Lower temperatures produce a slower rate of translation but often extend the time of the linear rate to several hours. Enhanced expression at lower temperatures for longer times appears to be gene/protein-specific and may be tried if the standard reaction at 37°C for 1 hour does not produce the desired results.

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Transcend™ Non-Radioactive Translation Detection System

Description

The Transcend™ Non-Radioactive Translation Detection Systems enable non-radioactive detection of proteins synthesized in vitro. Using this system, biotinylated lysine residues are incorporated into nascent proteins during translation, eliminating the need for labeling with [³⁵S]methionine or other radioactive amino acids. Biotinylated lysine is added to the translation reaction as a pre-charged ε-labeled biotinylated lysine-tRNA complex (Transcend™ tRNA) rather than a free amino acid. After SDS-PAGE and electroblotting, the biotinylated proteins can be visualized by binding either Streptavidin-Alkaline Phosphatase (Streptavidin-AP) or Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), followed either by colorimetric or chemiluminescent detection. Typically, 0.5–5ng of protein can be detected within 3–4 hours after gel electrophoresis. This sensitivity is equivalent to that achieved with [³⁵S]methionine incorporation and autoradiographic detection 6–12 hours after gel electrophoresis. For a detailed protocol and background information, please see Technical Bulletin #TB182

The use of Transcend™ tRNA offers several advantages

• No radioisotope handling, storage or disposal is needed.
• The biotin tag allows detection (0.5–5ng sensitivity).
• The biotin tag is stable for 12 months, both as the Transcend™ tRNA Reagent and within the labeled proteins. It is not necessary to periodically resynthesize biotin-labeled proteins, unlike ³⁵S-labeled proteins, whose label decays over time.
• Labeled proteins are detected as sharp gel bands, regardless of the intensity of labeling or amount loaded on the gel, thus allowing the detection of poorly expressed gene products.
• Results can be visualized quickly, using either colorimetric or chemiluminescent detection.

The precharged E. coli lysine tRNAs provided in this system have been chemically biotinylated at the ε-amino group using a modification of the methodology developed by Johnson et al. (1976). The biotin moiety is linked to lysine by a spacer arm, which greatly facilitates detection by avidin/streptavidin reagents (Figure 5.6). The resulting biotinylated lysine tRNA molecule (Transcend™ tRNA) can be used in either eukaryotic or prokaryotic in vitro translation systems such as the TNT^® Coupled Transcription/Translation Systems, Rabbit Reticulocyte Lysate, Wheat Germ Extract or E. coli S30 Extract (Kurzchalia et al. 1988). Lysine is one of the more frequently used amino acids. On average, lysine constitutes 6.6% of a protein’s amino acids, whereas methionine constitutes only 1.7% (Dayhoff, 1978).

Effects of Biotinylated Lysine Incorporation on Expression Levels and Enzyme Activity

Lysine residues are common in most proteins and usually are exposed at the aqueous-facing exterior. The presence of biotinylated lysines may or may not affect the function of the modified protein. In gel shift experiments, c-Jun synthesized in TNT^® Reticulocyte Lysate reactions and labeled with Transcend ™ tRNA performed identically to unlabeled c-Jun (Crowley et al. 1993).

Estimating Incorporation Levels of Biotinylated Lysine

Incorporation of radioactively labeled amino acids into proteins typically is quantitated as percent incorporation of the label added. This value can include incorporation of radioactivity into spurious gene products such as truncated polypeptides. Thus, percent incorporation values provide only a rough estimate of the amount of full-length protein synthesized and do not provide any information on translation fidelity. With Transcend™ tRNA reactions, it is difficult to directly determine the percent incorporation of biotinyl-lysines into a translated protein. An alternative means of estimating translation efficiency and fidelity in Transcend™ tRNA reactions is to determine the minimum amount of products detectable after SDS-PAGE. In all cases tested, we detected translation products in 1µl of a 50µl translation reaction using as little as 0.5µl of Transcend ™ tRNA (Figure 5.7). The amount of biotin incorporated increases linearly with the amount of Transcend™ tRNA added to the reaction, up to a maximum at approximately 2µl.

Capture of Biotinylated Proteins

Biotinylated proteins can be removed from the translation reaction using biotin-binding resins such as SoftLink™ Soft Release Monomeric Avidin Resin. Nascent proteins containing multiple biotins bind strongly to SoftLink™ Resin and cannot be eluted using “soft-release” nondenaturing conditions. SoftLink™ Resin is useful, however, as a substitute for immunoprecipitation.

Non-Radioactive Translation and Detection Protocol

Materials Required:

• Transcend™ Non-Radioactive Translation Detection System (Cat.# L5070, L5080)
• RNasin^® Ribonuclease Inhibitor (Cat.# N2111)
• Nuclease-Free Water (Cat.# P1193)
• translation extract
• salts, DTT and other components as needed to optimize translation reaction
• complete amino acid mix or a combination of two minus amino acid mixes
• PVDF or nitrocellulose membrane
• Tris-buffered saline (TBS)
• TBS + 0.5% Tween^® 20 (TBST)
• Optional: Ponceau S stain (Sigma Cat.# P7170)

Use the following protocol as a guideline for setting up translation reactions using Transcend™ tRNA. In general, Transcend™ tRNA may be used in any in vitro translation protocol at a concentration of 1µl Transcend™ tRNA per 50µl reaction. An example reaction using Rabbit Reticulocyte Lysate is provided.

The number of lysines in the translated polypeptide and the efficiency of translation are the two most important factors affecting band intensity of the translation product. To increase the intensity of weak bands, add up to double the standard amount of Transcend™ tRNA to the reaction (see Figure 5.7).

To reduce the chance of RNase contamination, wear gloves and use microcentrifuge tubes and pipette tips that have been handled only with gloves. Addition of RNasin^® Ribonuclease Inhibitor to the translation reaction is recommended but not required.

If the amount of translation product must be estimated, add radioactive amino acid(s) (in addition to Transcend™ tRNA) to either a control translation reaction or all translation reactions. Percent incorporation of the radioactive amino acid can be used in combination with knowledge of the protein’s amino acid composition to estimate the amount of translation product produced.

1. Thaw the Transcend™ tRNA on ice. Thaw the translation lysate by hand-warming and immediately place on ice. Thaw all other components at 37°C, and then store on ice.

2. Set up reactions on ice, adding all components except Transcend™ tRNA. Gently mix and briefly centrifuge if necessary. Add Transcend™ tRNA.

3. Immediately incubate the translation reaction at 30°C for 60 minutes.

4. Place the tube on ice to terminate the reaction.

Endogenous Biotinylated Proteins

Commonly used translation extracts contain endogenous biotinylated proteins, which may be detected when translation products are analyzed by SDS-PAGE, electroblotting and Streptavidin-AP detection. Rabbit Reticulocyte Lysate contains one biotinylated protein which migrates as a faint band at 100kDa and, in some lots, an additional very faint band at 47kDa. E. coli S30 Extract contains one endogenous protein, migrating at 22.5kDa. Wheat Germ Extract contains five major endogenous biotinylated proteins, migrating at 200kDa, 80kDa, 32kDa and a doublet at 17kDa. Comparison to a negative control reaction (without template) will distinguish endogenous biotinylated protein(s) from newly synthesized biotinylated translation product.

Optimization

Biotin labeling of poorly expressed proteins or proteins containing few lysines can be increased by doubling the amount of Transcend™ tRNA added per 50µl translation reaction (see Figure 5.7). For maximal expression, optimize the amount of template added to the reaction and use highly purified RNA or DNA.

Prokaryotic Coupled Transcription/Translation

In E. coli S30 reactions, increased band intensities often can be obtained by using 2–3 times the recommended DNA concentration.

Colorimetric and Chemiluminescent Detection of Translation Products

Biotin-containing translation product can be analyzed in either of two ways. The product can be resolved directly by SDS-PAGE, transferred to an appropriate membrane and detected by either a colorimetric or chemiluminescent reaction (Figure 5.8). Alternatively, biotinylated protein can be captured from the translation mix using a biotin-binding resin such as SoftLink™ Resin. This approach is useful as a replacement for immunoprecipitation of protein complexes.

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FluoroTect™ Green_Lys in vitro Translation Labeling System

Description

The FluoroTect™ Green_Lys in vitro Translation Labeling System uses a charged lysine tRNA molecule labeled with the fluorophore BODIPY^®-FL at the epsilon (ε) amino acid position of lysine (Figure 5.9). For the FluoroTect™ System, lysine was chosen as the labeled amino acid because it is one of the more frequently used amino acids, comprising, on average, 6.6% of a protein’s amino acids. Detection of the labeled proteins is accomplished in 2–5 minutes directly “in-gel” by use of a laser-based fluorescent gel scanner. This eliminates any requirement for protein gel manipulation such as fixing/drying or any safety, regulatory or waste disposal issues such as those associated with the use of radioactively labeled amino acids. The convenience of non-isotopic “in-gel” detection also avoids the time-consuming electroblotting and detection steps of conventional non-isotopic systems. For a detailed protocol and background information about this system, please see Technical Bulletin #TB285.

Translation Protocol

• FluoroTect™ Green_Lys in vitro Translation Labeling System (Cat.# L5001)
• Nuclease-Free Water (Cat.# P1193)
• translation system (e.g., TNT^® Coupled Transcription/Translation System, Rabbit Reticulocyte Lysate, Wheat Germ Extract or E. coli S30 Extract)
• complete amino acid mix or a combination of two minus amino acid mixes
• salts, DTT and other components as needed to optimize the translation reaction
• fluorescent imaging instrument (i.e., FluorImager^® SI or FluorImager^® 595 [Molecular Dynamics], both with a 488 argon laser; the Typhoon^® 8600 [Molecular Dynamics], with a 532nm excitation, or the FMBIO^® II [Hitachi], with a 505 channel)

Use the following protocol as a guideline for setting up translation reactions using FluoroTect™ Green_Lys tRNA. In general, FluoroTect™ Green_Lys tRNA may be used in an in vitro translation protocol at a concentration of 1µl of FluoroTect™ Green_Lys tRNA per 50µl reaction. Examples of standard reactions using TNT^® T7 Quick for PCR DNA and Rabbit Reticulocyte Treated Lysate are provided.

1. Assemble the following reactions on ice. Add all components except the FluoroTect™ Green_Lys tRNA, and gently mix by pipetting the reaction while stirring the reaction with the pipette tip. If necessary, spin briefly in a microcentrifuge to return the sample to the bottom of the tube. Add the FluoroTect™ Green_Lys tRNA.

2. Incubate at 30°C for 60–90 minutes (conditions will vary depending on the translation system used).

3. Terminate the reaction by placing on ice. If necessary, the translation reaction can be stored for several months at –20°C or –70°C.

Fluorescence Detection

The fluorescent translation product should be resolved by running a sample on an SDS-PAGE and then visualized by placing the gel on a laser-based fluorescence scanning device.

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References

1. Anderson, C. et al. (1983) Preparation of a cell-free protein-synthesizing system from wheat germ. Meth. Enzymol. 101, 635–44.
2. Andrews, D. (1987) Promega Notes 11.
3. Beckler, G. (1992) Optimization of in vitro translation reactions using the salt select lysate system. Promega Notes 35, 5–10.
4. Bocco, J.L. et al. (1988) Processing of SP1 precursor in a cell-free system from poly(A+) mRNA of human placenta. Mol. Biol. Reports 13, 45–51.
5. Chen, H. and Zubay, G. (1983) Prokaryotic coupled transcription-translation. Meth. Enzymol. 101, 674–90.
6. Chow, M. et al. (1992) Structure and biological effects of lipid modifications on proteins. Curr. Opin. Cell Biol. 4, 629–36.
7. Collins, J. (1979) Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3. Gene 6, 29–42.
8. Crowley, K.S. et al. (1993) The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–15.
9. Dayhoff, M.O. (1978) In: Atlas of Protein Sequence and Structure, Suppl. 2 National Biomedical Research Foundation, Washington, DC.
10. Ezure, T. et al. (2006) Cell-free protein synthesis system prepared from insect cells by freeze-thawing. Biotechnol. Prog. 22, 1570–7.
11. Glass, C.A. and Pollard, K.M. (1990) Promega Notes 26.
12. Gross, M., Rubino, M.S. and Starn, T.K. (1988) Regulation of protein synthesis in rabbit reticulocyte lysate. Glucose 6-phosphate is required to maintain the activity of eukaryotic initiation factor (eIF)-2B by a mechanism that is independent of the phosphorylation of eIF-2 alpha. J. Biol. Chem. 263, 12486–92.
13. Han K.-K. and Martinage, A. (1992) Post-translational chemical modification(s) of proteins. Int. J. Bichem. 24, 19–28.
14. Hanes, J. and Pluckthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–42.
15. Jackson, R. and Hunt, T. (1983) Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Meth. Enzymol. 96, 50–70.
16. Jackson, R.J. (1991) Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: Unique features of encephalomyocarditis virus RNA translation. Biochim. Biophys. Acta 1088, 345–58.
17. Jackson, R.J. and Standart, N. (1990) Do the poly(A) tail and 3´ untranslated region control mRNA translation? Cell 62, 15–24.
18. Johnson, A.E. et al. (1976) Nepsilon-acetyllysine transfer ribonucleic acid: A biologically active analogue of aminoacyl transfer ribonucleic acids. Biochem. 15, 569–75.
19. King, R.W. et al. (1997) Expression cloning in the test tube. Science 277, 973–4.
20. Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucl. Acids Res. 18, 2828.
21. Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–92.
22. Krieg, P. and Melton, D. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucl. Acids Res. 12, 7057–70
23. Kurzchalia, T.V. et al. (1988) tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products. Eur. J. Biochem. 172, 663–8.
24. Lesley, S.A. et al. (1991) Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies. J. Biol. Chem. 266, 2632–8.
25. Lustig, K.D. et al. (1997) Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Meth. Enzymol. 283, 83–99.
26. MacDonald, M.R. et al. (1988) Posttranslational processing of alpha-, beta-, and gamma-preprotachykinins. Cell-free translation and early posttranslational processing events. J. Biol. Chem. 263, 15176–83.
27. Mattheakis, L.C. et al. (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–6.
28. Noren, C.J. et al. (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. Science 244, 182–8.
29. Novac, O., Guenier, A.S. and Pelletier, J. (2004) Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucl. Acid Res. 32(3), 902–15.
30. Pelham, H.R.B. and Jackson, R.J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247–56.
31. Powell, S.M. et al. (1993) Molecular diagnosis of familial adenomatous polyposis. N. Eng. J. Med. 329, 1982–1987.
32. Pratt, J.M. (1984) In: Transcription and Translation, Hanes, B.D. and Higgins, S.J., eds., IRL Press, Oxford, UK.
33. Promega (1990) E. coli S30 Coupled Transcription Translation System. Promega Notes 26, 1–2.
34. Rando, R.R. (1996) Chemical biology of protein isoprenylation/methylation. Bichimica Biophysica Acta 1300, 5–16.
35. Ray, R.B. et al. (1995) Transcriptional regulation of cellular and viral promoters by the hepatitis C virus core protein. Virus Research 37, 209–20.
36. Roest, P.A. et al. (1993) Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum. Mol. Genet. 2, 1719–21.
37. Shyamala, V. and Ames, G.F. (1991) Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis. Gene 97, 1–6.
38. Snyder, M.J. and Edwards, R.D. (1991) Effects of polyamine analogs on the extent and fidelity of in vitro polypeptide synthesis. Biochem. Biophys. Res. Commun. 176, 1383–92.
39. Studier, F.W. and Moffatt, B.A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–30.
40. Suzuki, T. et al. (2006) Performance of expression vector, pTD1, in insect cell-free translation system. Biosci. Bioeng. 102, 69–71.
41. Thompson, D. and Beckler, G. (1992) TNT^® Lysate coupled transcription/translation: Comparison of the T3, T7 and SP6 Systems. Promega Notes 38, 13–7.
42. Walter, P. and Blobel, G. (1983) Preparation of microsomal membranes for cotranslational protein translocation. Meth. Enzymol. 96, 84–93.
43. Weber, L.A. et al. (1977) Inhibition of protein synthesis by Cl–. J. Biol. Chem. 252, 4007–10.
44. Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–87.
45. Zubay, G. (1980) The isolation and properties of CAP, the catabolite gene activator. Meth. Enzymol. 65, 856–77.

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