A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based
Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.
Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system.
The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate) templates into active proteins.
KeywordsPure System Rabbit Reticulocyte Lysate Reconstituted System Eukaryotic Ribosome Nascent Chain
an E. coli extract-based in vitro protein synthesis system
rabbit reticulocyte lysate or rabbit reticulocyte lysate-based in vitro protein synthesis system
S30 supplemented with RRL
- The PURE system
a reconstituted in vitro protein synthesis system based on protein translation in E. coli
green fluorescent protein
Cell-free protein synthesis has gained increasing popularity as a rapid and high throughput technology to obtain proteins from their genes [1, 2, 3]. Cell-free protein synthesis systems often use a cell lysate from E. coli cells, rabbit reticulocytes or wheat germ to supply the protein translation machinery and a recombinant T7 RNA polymerase to couple transcription to translation. Perhaps the biggest drawback of synthesizing proteins in the lysate is that the lysate contains a large portion of the cellular proteins and nucleic acids that are not necessarily involved in protein synthesis. How and whether these macromolecules affect the in vitro processes are largely unpredictable and often unknown. For instance, proteases and nucleases in the lysates could be inhibitory to protein synthesis. Cellular proteins or nucleic acids in the lysates may interfere with the functional assays and subsequent purification may be hampered by the low amount of the synthesized protein.
As a step closer to addressing these problems, protein translation was reconstituted in vitro from purified components of the E. coli translation machinery . Except for the ribosomes and tRNAs, which were purified from the E. coli lysate, this reconstituted system, appropriately named "the PURE system", contains purified recombinantly-expressed proteins of all E. coli translation factors and aminoacyl-tRNA synthetases . Remarkably, this reconstituted system has been shown to catalyze efficient in vitro protein synthesis . Largely free of other cellular components, the PURE system facilitates in vitro studies in a much cleaner background than a lysate-based system. The immediate impact of the PURE system and other similar reconstituted systems was their superior performance in such in vitro applications as the incorporation of unnatural amino acids , ribosome display [7, 8] and mRNA or pure translation display [9, 10], largely due to their designability and their significantly reduced nuclease and protease activities.
In spite of the advantages of the PURE system, we suspected that this "stripped-down" version of the protein translation machinery would encounter problems when "difficult" templates for translation were used. In this study, we compared protein synthesis of several proteins that exhibited significant differences when synthesized in the PURE system and an S30 system. An S30 system is an E. coli extract-based system, derived from the E. coli cell lysate obtained after 30,000 × g centrifugation . To begin to investigate the reasons for these differences, we added the size-separated fractions of an E. coli lysate to the in vitro reactions of the PURE system. Our data suggest the possibility of additional factors that further promote in vitro protein synthesis.
Many eukaryotic proteins that fold correctly in eukaryotes tend to misfold when expressed in E. coli. Such capability of eukaryotes, which has evolved to accommodate the needs for more complexity in proteins, has been attributed, at least in part, to the cooperativity of a large number of eukaryotic chaperones and their intimate association with translation and eukaryotic ribosomes . To investigate the differences in protein synthesis between prokaryotic and eukaryotic in vitro systems, we synthesized a fusion protein consisting of two eukaryotic protein domains, which was found to fold well in a rabbit reticulocyte system, but not in an E. coli system. In particular, we asked if this fusion protein translated by E. coli ribosomes would fold better in the presence of eukaryotic chaperones. Such experiments would be difficult to perform in vivo, as successful co-expression of a complete set of eukaryotic chaperones in E. coli is perhaps an impossible task by itself. We instead performed in vitro protein synthesis in the S30 system to which a eukaryotic lysate or its fractions were added. Our data illustrated striking differences in protein synthesis between prokaryotic and eukaryotic in vitro systems.
Results and discussion
Compared to the S30 system, the prokaryotic reconstituted system (the PURE system) produced noticeably less protein, and was more cold-sensitive
Significantly less protein and lower activity were observed in the PURE system than in S30 for Fluc and some other proteins (Fig. 1A &1C, B lane 1 and 3). The PURE system contains the minimal set of components necessary for in vitro protein synthesis, lacking numerous other factors that are involved in protein synthesis and nascent chain folding in vivo. A previous study showed that the major E. coli chaperones were largely absent in the PURE system, and addition of DnaK family chaperones and trigger factor to the PURE system enhanced the activity of a synthesized protein . It is possible that the presence of chaperones in S30 only accounted for the enhanced activity of already synthesized proteins, whereas the increase in the overall protein synthesis yield was due to other factors in S30.
Another interesting difference between the PURE system and S30 was that protein synthesis in S30 was less affected by low temperatures than in the PURE system. During the first 30 min. of protein synthesis, the difference in the initial accumulation of the activity (and the yield (not shown)) between 20°C and 37°C was much smaller in S30 than in the PURE system (Fig. 1C). At 20°C, almost no activity and protein (not shown) were observed during the first 30 min. of the synthesis in the PURE system (Fig. 1C). Even after 1 hr at 20°C, only a small amount of Fluc was detected in the PURE system (Fig. 1A). It is possible that this cold-sensitivity of the PURE system was due to its lack of certain cellular factors whose in vivo functions are to maintain efficient translation after cold shock .
Supplementing the PURE system with the E. colicell extract or its fractions improved the yield and activity to a level close to that of the S30 system
The SDS-PAGE gel analysis of the peak fractions suggested that the fractions 16 to 18 contained the bulk of the E. coli soluble proteins, consistent with their size range of ~10–150 kDa (Fig. 2). The data indicated that the "active ingredients" were likely to be macromolecules such as proteins, but not the small molecules, which were eluted at later fractions (data not shown). These "active" macromolecules could include those already present in the PURE system, such as translation factors, chaperones, and those lacking in the PURE system, such as other cellular factors that are important for efficient protein synthesis. Consistent with this notion, supplementing the PURE system reactions with F17 or Sup also significantly enhanced the protein synthesis yields at 20°C (data not shown). Since the PURE system is a coupled transcription/translation system, it is also possible that in vitro transcription was affected in such a way that protein translation was enhanced .
Supplementing the S30 system with the rabbit reticulocyte lysate or its fractions failed to produce an active full-length fusion protein
To investigate if the ability of RRL to synthesize the full-length FG was due to its translation or its folding machineries, S30 was supplemented with a crude rabbit reticulocyte lysate, resulting in a mixed system (S30+RRL). Cycloheximide, an antibiotic specific to the eukaryotic translation, was added to S30+RRL to prevent any residual translation from the eukaryotic ribosomes. S30+RRL was capable of synthesizing the full-length and active Fluc (Fig. 4A &4B). It appeared that in S30+RRL, protein translation occurred on the bacterial ribosomes, as the truncated Fluc product, seen only in S30, was also observed in S30+RRL (Fig. 4A, left panel). In addition, the protein synthesis in S30+RRL was sensitive to the inhibition by chloramphenicol, an antibiotic specific to prokaryotes (data not shown). It was apparent that the prokaryotic translation machinery was fully functional in S30+RRL, and under such conditions, the nascent chains from the E. coli ribosomes could be the substrates for folding by the eukaryotic chaperones present in the rabbit reticulocyte lysate supplement. However, when the fusion protein FG was synthesized in S30+RRL, no significant amount of the full-length FG and almost no Fluc activity were detected (Fig. 4A, middle panel, and 4B). This result remained the same whether the crude RRL (Pel-Freez) or the RRL from the in vitro protein synthesis kit (Promega), which presumably contained functional eukaryotic chaperones, was used as the supplement (data not shown). Lowering the temperatures (to 30°C or 23°C) to slow down the rate of translation from the E. coli ribosomes failed to make any difference in the case of FG in both S30 and S30+RRL (data not shown). In fact, the elongation rate of translation in S30 has been estimated to be similar to that of eukaryotic translation .
Western blot analyses of the in vitro synthesis reactions of FG in S30 or S30+RRL using anti-Fluc revealed a large number of products corresponded to a wide range of molecular weights shorter than the full length FG (Fig. 4C). These incomplete Fluc products were largely not seen in RRL (Fig. 4C). When anti-GFP was used to probe these products, only those with molecular weights larger than Fluc were detected, whereas those smaller than Fluc were not (Fig. 4C). These data suggest that these smaller truncated translation products were likely the result of premature termination or pausing during translation, not the degradation products of the synthesized full-length fusion proteins. Similar results were also observed by other studies .
Why did a large number of the elongating ribosomes in S30 or S30+RRL stop on the template of FG even before the full-length Fluc was synthesized (Fig. 4C), even though most ribosomes were capable of completing the synthesis on the templates of Fluc (Fig. 4A, left panel) and a fusion protein F-SH (Fig. 4A, right panel)? Since both the Fluc and GFP genes can be readily expressed on their own in S30 (Fig 1B), we speculate one possibility is that the sequence of GFP when fused to that of Fluc created a complex template, e.g., potential inhibitory structures on the mRNA template, or uniquely misfolded nascent chain, causing the E. coli ribosomes to pause or terminate prematurely, often even before the upstream Fluc domain was translated. The presence of eukaryotic chaperones or other factors has no effect as long as the translation was from the E. coli ribosomes (Fig. 4C). The eukaryotic ribosomes, perhaps with their associated chaperones and other factors, seemed to have the ability to overcome these obstacles.
In vitro protein synthesis was compared not only between the minimal PURE system and the extract-based system, but also between the prokaryotic and eukaryotic systems. The advantage of the in vitro approach allowed us to supplement one system with the lysates or their fractions from another system, thereby providing insights to the fundamental differences between these systems. Since ribosomes, translation factors and aminoacyl-tRNA synthetases are among the most conserved RNA and protein molecules, the PURE system may resemble an ancestral form of the minimal protein translation machinery, which, as this study suggests, is sufficient but not efficient in protein synthesis. The S30 system by comparison contains the modern bacterial version, with additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate) templates into active proteins.
The reagents for making the constructs of Fluc and the fusion proteins were from New England Biolabs. All antibodies against chaperones were purchased from StressGen Bioreagents (Ann Arbor, MI). Monoclonal anti-Fluc and anti-GFP antibodies, cycloheximide, and chloramphenicol were purchased from Sigma. The S30 system and the RRL system were purchased from Promega except when indicated otherwise. Other S30 systems used were Expressway (Invitrogen) and the RTS system (Roche biosciences). A crude rabbit reticulocyte lysate was purchased from Pel-Freez Biologicals (Rogers, AR). The PURE system (Classic II) was purchased from Post Genome Institute (Tokyo, Japan). 35S-methionine (37 Tbq/mmol) was from Amersham.
Plasmid constructs of the reporter proteins
The genes for firefly luciferase (61 kDa) (Fluc), Aequorea victoria GFP (27 kDa), and E. coli β-galactosidase (116 kDa) (lacZ) were cloned into a pUC19-derived vector containing a T7 promoter and a bacterial Shine-Dalgarno sequence. The vector for in vitro synthesis of E. coli dihydrofolate reductase (DHFR) was a control vector in the PURE system kit. The fusion protein FG (88 kDa) was constructed by fusing the N-terminus of GFP to the C-terminus of Fluc. Another fusion protein F-SH (81 kDa) consisted of the SH2–SH3 domain of human p120 (GAP) protein fused to the C-terminus of Fluc.
In vitroprotein synthesis reactions
All reactions were conducted following the recommended protocols by the manufacturers of the in vitro systems. All S30 systems were used in batch mode. 400–500 ng of the plasmid DNA was used for each 50 μl reaction. For 35S labeling, 4 μl 35S-methionine was added per 50 μl reaction. For the PURE system reactions containing the supplements, 2.5 μl of the S30 extract (Sup) or the concentrated fraction was used for each 25 μl reaction. The S30 extract was prepared from an exponentially growing E. coli strain according to the protocol of Zubay  and obtained from the laboratory of Chris Noren (New England Biolabs). For in vitro reactions in a mixed system (S30+RRL), 10 μl of the rabbit reticulocyte lysate from Pel-Freez or Promega and 2 ng/μl cycloheximide was added to the S30 reaction mixture in a final volume of 50 μl.
The Fluc activity was assayed using the Luciferase Assay System (Promega) and a microplate luminometer (Centro LB 640, Berthold Technologies). The same luminometer was also used to assay the lacZ activity in combination with the Galacto-Light™ system (Applied Biosystems).
Analysis of the synthesized proteins
Aliquots (1 μl for the western blot analyses or 5 μl for 35S labeling) were taken from the in vitro reactions and loaded on 10–20% Tris-glycine SDS PAGE gels (Invitrogen). The gels were either blotted for analyses with antibodies or dried for exposure to X-ray films.
For fractionation of the S30 extract, 1 ml of the extract (~12 mg/ml protein) in the column buffer (20 mM HEPES, pH 7.6, 50 mM KCl, 1 mM DTT) was loaded onto a Superdex 200 (10/300) column (Pharmacia). The fractions were collected and then concentrated 5–10 fold in Centricon tubes (Milipore). For fractionation of the crude rabbit reticulocyte lysate, 1 ml of the lysate in the RRL column buffer (30 mM HEPES, pH 7.2, 10 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, 25 mM KOAc, and 0.2 mM DTT) was loaded on the same column. The fractions were directly used for western blot analyses.
We thank Drs. Eric Cantor, Dongxian Yue and Chris Noren for helpful discussion and critical reading of the manuscript, and Yu Zheng for providing the lacZ gene. This study was supported by New England Biolabs and a NIH grant GM 073274.
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