The in vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications, including the rapid identification of gene products (e.g., proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies. The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. In principle, it should be possible to prepare a cell-free extract for in vitro translation of mRNAs from any type of cells. In practice, only a few cell-free systems have been developed for in vitro protein synthesis. In general, these systems are derived from cells engaged in a high rate of protein synthesis. This article will explain different approaches to in vitro protein synthesis (translation of purified RNA versus "linked" and "coupled" transcription:translation) and will also describe basic differences between eukaryotic and prokaryotic cell-free systems.
The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes, wheat germ and Escherichia coli. All are prepared as crude extracts containing all the macromolecular components (70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation and termination factors, etc.) required for translation of exogenous RNA. To ensure efficient translation, each extract must be supplemented with amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase for eukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for the E. coli lysate), and other co-factors (Mg2+, K+, etc.).
There are two approaches to in vitro protein synthesis based on the starting genetic material: RNA or DNA. Standard translation systems, such as reticulocyte lysates and wheat germ extracts, use RNA as a template; whereas "coupled" and "linked" systems start with DNA templates, which are transcribed into RNA then translated. Each of these systems is discussed below.
Rabbit Reticulocyte Lysate
Rabbit reticulocyte lysate is a highly efficient in vitro eukaryotic protein synthesis system used for translation of exogenous RNAs (either natural or generated in vitro). In vivo, reticulocytes are highly specialized cells primarily responsible for the synthesis of hemoglobin, which represents more than 90% of the protein made in the reticulocyte. These immature red cells have already lost their nuclei, but contain adequate mRNA, as well as complete translation machinery, for extensive globin synthesis. The endogenous globin mRNA can be eliminated by incubation with Ca2+-dependent micrococcal nuclease, which is later inactivated by chelation of the Ca2+ by EGTA. Ambion offers a nuclease-treated reticulocyte lysate. This type of lysate is the most widely used RNA-dependent cell-free system because of its low background and its efficient utilization of exogenous RNAs even at low concentrations (Figure 1). Exogenous proteins are synthesized at a rate close to that observed in intact reticulocyte cells.
Figure 1. Standard in Vitro Translation Procedure Using Rabbit Reticulocyte Lysate or Wheat Germ Extract.
Untreated reticulocyte lysate translates endogenous globin mRNA, exogenous RNAs, or both. This type of lysate is typically used for studying the translation machinery, e.g. studying the effects of inhibitors on globin translation. Both the untreated and treated rabbit reticulocyte lysates have low nuclease activity and are capable of synthesizing a large amount of full-length product. Both lysates are appropriate for the synthesis of larger proteins from either capped or uncapped RNAs (eukaryotic or viral).
Wheat germ extract is a convenient alternative to the rabbit reticulocyte lysate cell-free system. This extract has low background incorporation due to its low level of endogenous mRNA. Wheat germ lysate efficiently translates exogenous RNA from a variety of different organisms, from viruses and yeast to higher plants and mammals. The wheat germ extract is recommended for translation of RNA containing small fragments of double-stranded RNA or oxidized thiols, which are inhibitory to the rabbit reticulocyte lysate. Both retic and wheat germ extracts translate RNA isolated from cells and tissue or those generated by in vitro transcription (see Figure 1). When using RNA synthesized in vitro, the presence of a 5' cap structure may enhance translational activity. Typically, translation by wheat germ extracts is more cap-dependent than translation by retic extracts. If capping of the RNA is impossible and the protein yield from an uncapped mRNA is low, the coding sequence can be subcloned into a prokaryotic vector and expressed directly from a DNA template in an E.coli cell-free system.
E. coli cell-free systems consist of a crude extract that is rich in endogenous mRNA. The extract is incubated during preparation so that this endogenous mRNA is translated and subsequently degraded. Because the levels of endogenous mRNA in the prepared lysate is low, the exogenous product is easily identified. In comparison to eukaryotic systems, the E.coli extract has a relatively simple translational apparatus with less complicated control at the initiation level, allowing this system to be very efficient in protein synthesis. Bacterial extracts are often unsuitable for translation of RNA, because exogenous RNA is rapidly degraded by endogenous nucleases. There are some viral mRNAs (TMV, STNV, and MS2) that translate efficiently, because they are somewhat resistant to nuclease activity and contain stable secondary structure. However, E.coli extracts are ideal for coupled transcription:translation from DNA templates.
In standard translation reactions, purified RNA is used as a template for translation. "Linked" and "coupled" systems, on the other hand, use DNA as a template. RNA is transcribed from the DNA and subsequently translated without any purification. Such systems typically combine a prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts to synthesize proteins from exogenous DNA templates. DNA templates for transcription:translation reactions may be cloned into plasmid vectors or generated by PCR (Primer Sequences for PCR-generated Translation Templates).
The "linked" system is a two-step reaction, based on transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate (Figure 2). Because the transcription and translation reactions are separate, each can be optimized to ensure that both are functioning at their full potential. Conversely, many commercially available eukaryotic coupled transcription:translation systems have compromised one or both reactions so that they can occur in a single tube. Thus, yield is sacrificed for convenience.
Figure 2. Linked in Vitro Transcription and Translation Procedure Using Rabbit Reticulocyte Lysate.
Unlike eukaryotic systems where transcription and translation occur sequentially, in E. coli, transcription and translation occur simultaneously within the cell. In vitro E. coli translation systems are thus performed the same way, coupled, in the same tube under the same reaction conditions (one-step reaction; Figure 3). During transcription, the 5' end of the RNA becomes available for ribosomal binding and undergoes translation while its 3' end is still being transcribed. This early binding of ribosomes to the RNA maintains transcript stability and promotes efficient translation. This bacterial translation system gives efficient expression of either prokaryotic or eukaryotic gene products in a short amount of time. For the highest protein yield and the best initiation fidelity, make sure the DNA template has a Shine-Dalgarno ribosome binding site upstream of the initiator codon. Capping of eukaryotic RNA is not required. Use of E.coli extract also eliminates cross-reactivity or other problems associated with endogenous proteins in eukaryotic lysates. Also, the E. coli S30 extract system allows expression from DNA vectors containing natural E. coli promoter sequences (such as lac or tac).
Figure 3. Coupled in Vitro Transcription:Translation Procedure Using E. coli Extract.
There are some significant differences between prokaryotic and eukaryotic mRNA transcripts. Typically, eukaryotic mRNAs are characterized by two post-transcriptional modifications: a 5'-7 methyl-GTP cap and a 3' poly(A) tail. Both modifications contribute to the stability of the mRNA by preventing degradation. Additionally, the 5' cap structure enhances the translation of mRNA by helping to bind the eukaryotic ribosome and assuring recognition of the proper AUG initiator codon. This function may vary with the translation system and with the specific mRNA being synthesized. The consensus sequence 5'-GCCACCAUGG-3', also known as the "Kozak" sequence, is considered to be the strongest ribosomal binding signal in eukaryotic mRNA. For efficient translation initiation, the key elements are the G residue at the +1 position and the A residue at the -3 position. An mRNA that lacks the Kozak consensus sequence may be translated efficiently in eukaryotic cell-free systems if it possesses a moderately long 5'-untranslated region (UTR) that lacks stable secondary structure.
In bacteria, the ribosome is guided to the AUG initiation site by a purine-rich region called the Shine-Dalgarno (SD) sequence. This sequence is complementary to the 3' end of the 16s rRNA in the 30S ribosomal subunit. Upstream from the initiation AUG codon, the SD region has the consensus sequence 5'-UAAGGAGGUGA-3'. Specific mRNAs vary considerably in the number of nucleotides that complement the anti-Shine-Dalgarno sequence of 16S rRNA, ranging from as few as two to nine or more. The position of the ribosome binding site (RBS) in relation to the AUG initiator is very important for efficiency of translation (usually from -6 to -10 relative to the A of the initiation site).
See "Ribosomal Binding Sites Sequence Requirements" for more information.
DNA templates for translation using "coupled" or "linked" transcription:translation systems can be easily generated by PCR. Below are the upstream (5')primer sequences to produce PCR products for T7-driven transcription and subsequent translation in a retic lysate and E.coli extract, respectively. Note, translation systems that use T3 or SP6 polymerases are also available . To generate PCR templates for other polymerases, simply change the T7 Promoter Sequence to a T3 or an SP6 sequence.