Cell-Free And Happy: In Vitro Translation And Transcription/Translation Systems

Date: June 21, 1999Kits for In Vitro Translation and Transcription/Translation Systems Problem proteins--you know the ones. Some are rapidly degraded by endogenous proteins. Others are toxic to the very cells used to overexpress them. These types of problems can sometimes be avoided by using cell-free extracts for the in vitro expression of proteins. In vitro translation systems are also useful for other applications, including incorporating modified or unnatural amino acids for functional studies and localizing mutations via synthesis of truncated gene products. In "conventional" systems, RNA is used directly as a template for translation. The RNA can be total RNA, mRNA, or a synthesized template. "Linked" or "coupled" translation systems start off with a DNA template. RNA is transcribed from the DNA template, then subsequently translated without purification. Appropriate synthetic DNA templates for in vitro transcription/translation systems are readily generated, either after cloning into plasmid vectors or via PCR. Coding sequences are generally preceded by one of several bacteriophage promoters (T7, T3, or SP6) and translation control elements specific for the chosen prokaryotic or eukaryotic cell-free lysate system. Image supplied by Ambion In vitro translation In coupled systems, transcription and translation occur in the same tube. Kits utilizing both prokaryotic and eukaryotic lysate systems are commercially available. Transcription and translation occur simultaneously within prokaryotic cells. During transcription, the 5' end of RNA is available for ribosomal binding. Translation commences while the 3' end is still being transcribed. This early RNA/ ribosome interaction stabilizes transcripts and promotes efficient translation. Kits that exploit the "naturally" coupled bacterial transcription/translation reactions are on the market. In eukaryotes, by contrast, transcription and translation occur in separate compartments at different times. Conditions that allow both transcription and eukaryotic translation to occur in a single tube may be suboptimal for one or both reactions. However, eukaryotic coupled transcription/translation kits have been successfully used by many researchers for numerous applications, and thus represent a viable option. Linked systems are two-step procedures. First, transcription is catalyzed with an appropriate polymerase. Second, a cell-free lysate system, usually eukaryotic, is used to effect translation. Because the transcription and translation reactions are separate in linked systems, each can be optimized. Whether planning to use a conventional, linked, or coupled system, you must first consider which type of translation lysate to employ. The most frequently used cell-free translation systems consist of crude extracts from E. coli, wheat germ, or rabbit reticulocytes along with supplements required for efficient translation. The cell-free extracts contain ribosomes, aminoacyl-tRNA synthetases, along with all of the other requisite macromolecular components and factors for the translation of RNA. These extracts are generally supplemented with amino acids, energy sources, energy regeneration systems, and various cofactors. In vitro translation systems based on rabbit reticulocyte lysates are highly efficient. Reticulocytes are highly specialized cells primarily responsible for the synthesis of hemoglobin, which comprises the bulk of the protein produced by these cells. The endogenous globin mRNA can be eliminated by incubation with a Ca2+-dependent nuclease. The nuclease is later inactivated via addition of the chelator EGTA. Nuclease-treated retic lysate is the most widely used cell-free system for translating RNA templates. This system is popular because of its low background and its efficient utilization of exogenous RNAs, even at low concentrations. Untreated retic lysates are primarily used to study the translation machinery itself. (For example, one can monitor the effects of exogenous compounds on globin translation.) Retic lysates have low nuclease activity and are capable of synthesizing large amounts of full-length products. Either capped or uncapped RNAs from viral or eukaryotic sources can be translated. Image supplied by Ambion Translation procedure Wheat germ lysates efficiently translate exogenous RNAs from a wide variety of organisms. These systems enjoy low background incorporation due to low levels of endogenous mRNA. Wheat germ systems are generally more cap dependent than are retic systems. Wheat germ systems are often preferable when synthesizing relatively small proteins (12-15 kDa) that comigrate with globin, which is abundant in reticulocyte extracts. You might also want to consider using a wheat germ-based method when translating mRNA coding for regulatory factors present at high levels in mammalian reticulocytes but not in plant extracts. It is also worthwhile to evaluate wheat germ systems for the translation of templates that are, for whatever reason, transcribed poorly in reticulocyte extracts. E. coli's relatively simple translational apparatus and less complicated initiation control mechanisms make extracts from these bugs particularly attractive for protein synthesis applications. But there is a catch. Bacterial extracts contain nucleases that rapidly degrade most exogenous RNAs. Some nonbacterial mRNAs are resistant to nuclease digestion. For example, mRNAs from TMV and several other viruses can be efficiently translated in cell-free extracts from E. coli. As discussed above, E. coli extract-based coupled transcription/translation kits have been developed. These procedures can use DNA templates corresponding to coding sequences from any species. Naturally occurring mRNAs from eukaryotes and many viruses are "capped" with 7-methylguanosine at their 5' termini. Capped mRNAs are generated in vitro by including an analog of the cap structure in the transcription reaction. The cap improves mRNA stability and should generally be included in templates that are to be injected in living cells. The 5' cap also increases the amount of proteins synthesized in wheat germ extracts. Thus, it is generally advisable to cap RNA templates prior to conventional in vitro translation via wheat germ systems. The cap is not usually necessary for efficient translation of RNA in reticulocyte systems. Most in vitro translation reactions are followed by the incorporation of a radiolabeled amino acid (typically [35S]-methionine) into the expressed product. Nonisotopic modified amino acids can also be incorporated and measured. For example, some companies offer kits for the incorporation of biotinylated lysine into the product. Biotin-labeled polypeptides can subsequently be detected via binding to avidin-labeled reagents. If appropriate antibodies are available, synthesis of the protein of interest may be monitored via Western blot analyses. If the protein of interest has a measurable biological activity, it may be assayed directly. Ambion's PROTEINscript-PRO™ kit for coupled transcription/translation differs from coupled kits offered by other vendors in that it uses an E. coli extract. It can be used to translate both prokaryotic and eukaryotic sequences. Ambion reports that yields can be even higher than those enjoyed using reticulocyte translation (100-1,000 ng per 50 µl reaction). Circular or linear DNA templates are generally used with this kit. Certain viral mRNAs (TMV, STNV, or MS2) can also be translated with this E. coli-based system. The company also offers PROTEINscript II™, a eukaryotic in vitro translation system in which transcription and translation are linked but occur as separate steps. Each reaction is individually optimized to generate the maximum yield of full-length protein. You only need to add DNA template, water, and [35S]-methionine. The company states that yields are typically 50-200 ng per 50 µl reaction. Comparable yields are obtained using Ambion's conventional kits for the translation of RNA templates with retic (50-200 ng) or wheat germ (40-160 ng) lysates. Ambion's TechNotes 5(5) provides more information about some of these products. This issue also contains W. A. Kudlicki and A. Coffman's well-written article that summarizes the basics of in vitro transcription/translation. ;The Single Tube Protein™ System 3 (STP3) from Novagen is a linked, rabbit reticulocyte-based kit. This system is designed for the synthesis of proteins from circular or linear DNA templates containing a bacteriophage T7 or SP6 promoter. Also available are STP3-Biotin kits, which include biotin-tRNAlysine. Adding this modified tRNA to the translation reaction enables the incorporation of biotin into the target polypeptide at lysine residues. The company recommends using a member of one of its two different vector product lines for template production. Novagen states that its pCITE (U.S. Patent No. 4,937,190) vector carries an enhancer that increases translation efficiency by five- to 10-fold. pCITE vectors that add sequences encoding amino terminal S*Tags are also available. S*Tags can be assayed spectrophotometrically or detected using Western blot analyses. The spectrophotometric assay measures ribonuclease S activity. This enzyme assay is based on the binding of S*Tag peptides and S proteins to form the active ribonuclease complexes. Antibodies specific for the S*Tag peptide enable detection via Western blot analyses or other immunochemical methods. The tags can also be used to affinity-purify the expressed protein. Novagen also says that its pT7-Blue-2 plasmid is designed for optimal performance with its STP3 system. In addition to an enhancer element, this vector has an S*Tag sequence. As the name implies, it enables blue/white screening for identification of clones bearing the template of interest. Novagen's Primer to Protein System 3 uses STP3 reagents along with elements from a cloning kit to effect the transcription/translation of PCR products bearing open reading frames. Also, kits optimized for the conventional translation of capped versus noncapped RNA transcripts are available. Its SpeedRead™ Lysates are designed with convenience in mind. The researcher simply adds radiolabeled methionine, water, and RNA template to the mixes. The Red Nova® Lysate can be used for labeling with methionine, cysteine, or leucine. The company states that Red Nova® is more concentrated than other commercially available preparations, enabling the use of more dilute RNA templates. Image supplied by Roche Molecular Biochemicals Principle of biotin in vitro translation and chemiluminescent detection Amersham Pharmacia Biotech's rabbit reticulocyte lysate system is quality controlled with 3H, 14C, 35S labeled leucine, methionine, and cysteine respectively. The positive control mRNA generates a protein with intrinsic autocleavage activity generating a molecular weight marker of 73, 51, 30, 22, and 6 kDa respectively. AP Biotech also offers a nonradioactive in vitro translation system that couples detection with its ECL detection system via Streptavidin HRP. The nonrad system also contains a bifunctional control mRNA yielding a molecular weight marker of 20.35, 94, and 106 kDa. AP Biotech's ECL cell-free labeling systems can be combined with any in vitro translation or transcription/translation system, whereas the ECL in vitro translation systems come with rabbit reticulocyte lysate. A coupled in vitro transcription/translation protocol is available for the company's in vitro translation/transcription products. Roche Molecular Biochemicals vends nonradioactive and radioactive versions of its Linked in vitro SP6/T7 Transcription/Translation kit. The nonradioactive kit is based on the incorporation of biotin-labeled lysine into polypeptides, and the radioactive counterpart is designed for use with [35S]-methionine. These kits were designed for the time-efficient production of small quantities of protein from DNA templates. The radioactivity-based procedure takes less than 3 hours to perform. Processing samples using the nonradioactive kit takes about 6 hours. The company offers two different reticulocyte translation kits. Gel filtration has been used to deplete the endogenous amino acids from lysates included in its "Translation Kit, Reticulocyte, Type I." This treatment enables higher incorporation of radiolabeled amino acids. Endogenous amino acids have not been removed in the less costly "Translation Kit, Reticulocyte, Type II." Roche recommends the less expensive kit for routine assays of mRNA translation efficiencies. Promega offers a versatile line of translation products. Both wheat germ-and rabbit reticulocyte-based versions of its TNT® Coupled Transcription/Translation Systems are available. Promega has compiled an impressive bibliography (Part # BL001) listing many references that cite usage of its TNT® products. The E. coli Extract for Linear Templates can be used for the coupled transcription/translation of different template types, including DNA fragments, PCR-synthesized DNA and prokaryotic and synthetic RNA. Promega also offers a kit for the coupled transcription/translation of circular DNA. Systems for the translation of RNA templates utilizing extracts from wheat germ, retic, or E. coli are also offered. The Flexi® Rabbit Reticulocyte Lysate System enables the fine-tuning of Mg2+, K+, and dithiothreitol concentrations for the optimal translation of given mRNAs. Sigma's in vitro Translation System for the translation of RNA templates with rabbit reticulocyte lysates is supplied with four different labeling mixes. Two of the mixes are used to label capped mRNA with either methionine or leucine. The other two mixes are for labeling the uncapped counterparts with the same amino acids. Stratagene claims that its reticulocyte-based In Vitro Express™ Translation Kit allows more incorporation of [35S]-methionine than do other commercially available lysates. Amersham Pharmacia Biotech sells retic translation kits with or without biotin-tRNAlysine. Its ECL in vitro Translation Systems for generating biotinylated proteins is available with or without 2,000 cm2 membrane for Western blot analyses. Contact information for companies that vend in vitro translation and transcription/translation kits is summarized in the table. Kits designed exclusively for transcription are also available from many of these companies. However, you should be aware that in vitro transcription kits optimized for numerous other applications are on the market, such as in vitro transcription kits for antisense/microinjection studies and for the isolation of RNA binding proteins. Many companies sell kits optimized for the generation of high specific activity radiolabeled RNA probes needed for Northern blot analyses. Other vendors mentioned in this article offer kits geared toward more specific applications, such as protein truncation test (PTT) kits for the detection of mutations causing the premature termination of proteins. The author can be contacted at deborahwilkinson@sprynet.com. Kits for In Vitro Translation and Transcription/Translation Systems

Cell-Free And Happy: In Vitro Translation And Transcription/Translation Systems

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