The extract from washed wheat embryos provides a powerful and versatile method for cell-free protein synthesis based on the accumulated DNA sequence i... Purification - Control system synthesis - Data mining - Embryo - Materials science and technology - Protein engineering - Synthetic biology - Fractionation - Protocols - Milling machines - chemical reactions - DNA - genetics - proteins - plant translation factors - washed wheat embryos - cell-free protein synthesis - DNA sequence information - translation reaction - eukaryotic protein synthesis - eukaryotic elongation factors
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Purifi'cation of Plant Translation Factors for Reconstitution of Protein Synthesis
Hikaru Nagano, Shouhei Sugihara, Hisanori Takagi, Tomio Ogasawara, Yaeta Endo, and Kazuyuki Takai Department of Material Science and Biotechnology, Graduate School of Science and Engineering, Venture Business Laboratory, and Cell-free Science and Technology Research Center, Ehime University 3, Bunkyo-cho, Matsuyama, Ehime 790-8577 Japan
Abstract: The extract from washed wheat embryos provides a powerful and versatile method for cell-free protein synthesis based on the accumulated DNA sequence information. Although the extract contains everything needed for protein synthesis, some of the required components may be uncharacterized, and some components of the extract do not directly involve in, or even inhibit protein synthesis. This has hampered us from detailed analyses of the translation reaction. Therefore, we have decided to make an attempt to construct a reconstituted protein synthesis system, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and its regulation and would probably be applicable to synthetic biology and artificial manipulation of the translational apparatus. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryotic elongation factors (eEFs) IA, iB, and 2. eEFIA and eEF2 supported polyphenylalanine synthesis.
embryos that are free from endosperm materials, the ribosomes and the translation apparatus are quite robust during long incubation periods12. Thus, wheat may be the most promising source that could be used for the investigation of cellular regulation mechanisms mediated by the translation machinery by constitutive or synthetic biology approaches. However, the cell extract used for the wheat cell-free translation contains unknown molecules and those that are not involved in protein synthesis. It also contains mRNA degradation enzymes that complicate the protein synthesis reaction. In addition, understanding of the mechanisms in eukaryotic protein synthesis reactions, including initiation, elongation, termination, polysome formation and disassembly, protein folding and subunit joining, and so on, is still incomplete. Therefore, we decided to try reconstitution of protein synthesis based on the apparatus from wheat. As the first step, in the present study, we followed the protocols established more than 20 years ago5 for purifying elongation factors.
2.1 The S30 fraction from crude wheat germ Crude wheat germ for preparation of protein factors was a custom-order product from Kawakubo Flour Mill Co., Matsuyama, Japan. It was unroasted and undried after sieving, and was stored at -20°C in aliquots before use. In order to prepare extracts from this crude wheat germ fraction, it was homogenized essentially as described5: The wheat germ fraction was ground to fine powder with a Zojirushi BM-FX08-GA Mill Mixer. To 8 to 9 grams of the powder, Buffer B (20 mM HEPES-KOH, pH 7.6, 1 mM magnesium acetate, 2 mM calcium acetate, 6 mM 2-mercaptoethanol, 120 mM potassium chloride, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) was added up to 35 ml, and the mixture was vortexed to dissolve the soluble materials as well as possible. The mixture was centrifuged at 14,170 g at 4°C for 30 min, and the aqueous supematant was recovered so that the oil layer was not included. This supematant was centrifuged at 30,000 g at 4°C for 20 min, and the supematant (S30) was recovered and was used for further fractionation.
After the genome sequencing projects, elucidation of the interactions of the gene products that constitute complex networks supporting all activities of the cells is a major issue for understanding life. In particular, eukaryotic cells have developed very complex regulation systems that may cause differentiation, cancer, cell death, and so on. The wheat cell-free protein synthesis system from washed embryo particles13 is a very powerful tool for preparation of proteins used for experimental approaches to the understanding of protein networks. It provides methods for preparing many different proteins in parallel in a short period of time. It has also been shown that multi-domain proteins, which are more dominant in eukaryotic cells than in prokaryotic cells, tend to be produced in soluble forms in the wheat cell-free synthesis system4. Therefore, the wheat cell-free system may be very useful for reconstitution of eukaryotic cellular processes supported by many different proteins. It is noteworthy that many of these cellular regulation events are controlled to some extent through the regulation of protein synthesis, especially in eukaryotes. In order to understand these processes by a constitutive approach, a protein synthesis system made exclusively of well-characterized components. As the protein synthesizing S30 extract is derived from extensively washed wheat
2.2 The S30 fraction from washed wheat embryo particles and the ribosomes The S30 fraction from washed wheat embryo particles, which was used as the source of the ribosomes, was prepared
as described'. The ribosomes were purified by a standard sucrose density gradient method and was finally suspended in a buffer containing 35 mM Tris-HCl, 9 mM magnesium acetate, 70 mM potassium chloride, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol.
2.3 Aminoacyl-tRNA protection assay Aminoacyl-tRNA protection assay for the determination of the activities in the eEFIA fraction was essentially performed as described6, with the use of a transcript of a variant of serine tRNA from E. co1i7: [3H]-Ser-tRNA was prepared as described with the use of the crude Ser-tRNA synthetase crude fraction7. The eEF1A fraction was incubated at 30°C for 10 min in a buffer containing 75 mM HEPES-KOH, pH 8.0, 75 mM ammonium chloride, 15 mM magnesium chloride, 7.5 mM dithiothreitol (DTT), 60 bovine serum albumin (BSA), 0.1 mM GTP, 2.4 mM phosphoenolpyruvate, and 2.5 unit/ml pyruvate kinase (Sigma), to which [3H]Ser-tRNA (28,000 cpm) was added in a 50-pl solution at 30°C, and a 10-pl aliquot was spotted onto a slip of filter paper at each time point. The filter slips were rinsed with ice-cold 5% trichloroacetic acid solution three times to remove released [3H]Ser, and were dried. These samples were counted in a liquid scintillation counter.
The S30 fraction from the crude wheat germ fraction was fractionated essentially according to the literature5. 200 ml of S30 was centrifuged at 170,000 g for 3.5 h, and the supematant was fractionated with ammonium sulfate (AS). The 40-70%O AS fraction was obtained and was further fractionated on a DE52 column preequilibrated with a buffer containing 40 mM potassium chloride (Figure 1). We eluted the sample with the step salt gradient shown in the figure, while the original authors5 performed the chromatography with linear gradients. We found that eEFIA and eEFIB are eluted at the expected salt concentration ranges. However, we found that eEF2 distributed over a very wide range. KCI (mM)
2.4 Poly(U)-dependent poly(Phe) synthesis Poly(Phe) synthesis was performed essentially as described5 with some modification. For the preparation of [l4C]Phe-tRNAPhe, the wheat-germ S30 extract (from the crude germ fraction) was first passed through a column of DE52 (Whatman) to remove nucleic acid. This crude
enzyme fraction was then incubated with ['4C]Phe and yeast
tRNAPhe (Sigma), and the aminoacyl-tRNA was extracted
from acidic phenol and precipitated with ethanol. The S78 fraction was prepared by ultracentrifugation of the S30 fraction at 78,000 g for 3.5 hr. The supematant was stored after addition of 1 mM DTT, and the buffer was exchanged before use with 24 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.5 mM magnesium acetate, 0.4 mM spermidine, 2 mM DTT, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate (1 xDB) with the use of a Microcon YM50 (Amicon). For poly(Phe) synthesis, samples were incubated in a 25-pl reaction mixture containing 1 xDB plus 1 ptg/ml creatine kinase, 80 ptg/ml poly(U) , and 6,700 cpm ['4C]Phe-tRNA. A 5-pl-aliquot was transferred to a sodium hydroxide solution to stop the reaction and hydrolyze unused aminoacyl-tRNA. The samples were spotted on slips of filter paper and were rinsed with acid solution and counted as above. 3. RESULTS 3.1 Fractionation of the S30 extract on a DE52 column
Figure l. DE52 chromatography separating the 40-70 AS
fraction from the S170 supematant. The dimension of the column was 2.5 cm x 50 cm, and the buffer contained 20 mM HEPES-KOH, pH 7.6, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and potassium chloride. Each fraction tube contained about 13 ml of the eluate.
3.2 Purification of eEFIA The fraction containing eEFIA (92 ml) was fractionated by ammonium sulfate precipitation (the 60-80% AS fraction was collected) and was further separated on a 1.5 x 20 cm Whatman P11 column preequilibrated with a buffer containing 100 mM potassium chloride (Figure 2). The fractions of the highest purity as judged by the electrophoresis results (#53-60) and that of the modest purity (#50-52) were tested for their aminoacyl-tRNA hydrolysis protection activities (Figure 3). The results showed that these preparations could bind aminoacyl-tRNAs.
3.3 Purification of eEF2 and poly(Phe) synthesis The eEF2 fraction was collected and was applied to another P11 column. eEF2 eluted at a low salt range but did not make a sharp peak in this column chromatography, either. The purity of this eEF2 fraction was low (data not shown). Nevertheless, the eEF2 fraction obtained by this way could be used for supporting poly(U)-dependent
poly(Phe) synthesis (Figure 4). Although the reaction without the addition of the eEF2 fraction also showed low amino acid incorporation, the effect of the eEF2 fraction was clear.
| 1 0
Figure 3. The eEFlA fractions obtained.
(a) An SDS polyacrylamide gel separating the eEFIA fractions. M, marker; 1, fraction #50-52; 2, fraction #53-60. The arrow shows the
position of eEFIA. (b) Aminoacyl-tRNA retains after the indicated period during incubation with fraction #50-52 (filled triangles) or fraction # 53-60 (open squares), or without eEFIA (filled squares).
The embryo particles prepared by sieving of the crushed wheat seeds contain white matter originated from endosperm. This white matter contains tritin9, the ribosome inactivating protein (RIP) of wheat, which catalyzes depurination at the ricin-sarcin loop (SRL) of the ribosomes. It was believed in
l. . . .WX. . . r. g=. .
..the wheat ribosomes'. However, it was finally revealed in ~~~~~~~~2000 .that, by washing out the white matter from the embryo ~~~~~~~~particles beftore hlomogenization, a very effitcient and robust cell-free translation extract could be prepared'. The
ribosomes and other translation factors are inherently very
uiiaino E A
The eEFlA fraction from the DE52 column was fractionated by
ammonium sulfate and separated on a column of P11t. (a) The chromatogram. (b) SDS-polyacrylamide gels separating the fractions.
Fractionation of wheat germ extract for reconstitution of eukaryotic protein synthesis was challenged in early 1980's8. At that time, the knowledge about the mechanisms of eukaryotic translation was much poorer than today, and the techniques for purification of proteins were less developed. Even though, the reconstitution of wheat translation was possible by assembling a dozen different fractions from wheat germ. The efficiencies in such reconstituted translation system should have been much lower than those in the current wheat cell-free protein synthesis systems: it was found that the low efficiency and stability associated with conventional wheat germ cell-free translation systems originated from the low purity of the wheat germ
Reconstitution of protein synthesis was already accomplished with the ribosomes and other factors from E.
coil. The reconstituted translation system is available from a commercial source and is called "PURE System". Each of the protein factors in the PURE System has a histidine tag and has been overexpressed in E. coili and purified. The method for purification of the ribosomes was recently improved significantly'2, and the applications of the PURE System are growing because it is useful for analyses of the mechanisms in protein synthesis and for synthetic biology. Now that we have much sequence information concerning eukaryotic cells, it is time to try to reconstitute a eukaryotic protein synthesis system for the use in the analyses of the mechanisms in eukaryotic translation and synthetic biology. Wheat may be one of the most promising sources of the translation apparatus, because the embryo particles could be isolated from other parts ofthe plant: mammalian organisms, such as liver, are in general a mixture of differently differentiated cells, and it is possible that the ribosomes within such samples may be heterogeneous. It is well known that the wheat ribosomes could work for more than 2 weeks in the cell-free translation system if mRNA is supplied properly" 2. Therefore, we started to make efforts to
reconstitute protein synthesis with the wheat ribosomes. In order to reconstitute protein synthesis it is necessary to purify each of the many components that support protein synthesis. Let us classify the components: (i) the ribosomes, protein factors involved in (ii) initiation, (iii) elongation, (iv) termination, and (v) polysome formation, (vi) tRNA, (vii) aminoacyl-tRNA synthetases, (viii) proteins that support correct protein folding, membrane translocation, post-translational processing, and mRNA degradation. Although category (viii) is very important for the purpose of elucidating mechanisms supporting cellular functions, it is far beyond the scope of this paper because folding and the other functions could only be studied when the other components corresponding to category (i) to (vii) could work properly. Out of these seven, tRNA could be purified easily, and the elongation factors were well known. It is also necessary to determine the activities of the components for the purpose of preparation. Therefore, we started with purification of the elongation factors by conventional methods. The activities of the elongation factors could be determined by conventional methods, such as poly(U)-dependent poly(Phe) synthesis.
8000 G X)
chromatography. It is possible that eEF2 in our wheat germ is heterogeneous because the seeds originated from different areas of production. However, our preliminary data show that it is possible to recover this protein in a much sharper peak by the use of more recent chromatography media (data not shown). We are now trying to improve the methods for purification of these elongation factors by using more recent chromatography media. Hydrophobic interaction chromatography is one of the most promising because it was not widely used in 1980's and provides a different mode of separation. We have also begun to test if specific translation initiation supported by an internal ribosome entry site (IRES) or some leaderless mRNAs could be detected in a mixture containing the ribosomes, eEFIA, and eEF2, and aminoacyl-tRNA. This would accelerate reconstitution of protein synthesis, if possible: an IRES from cricket paralysis virus has been reported to bypass eIF-dependent initiation process in a mammalian system'3, and some leaderless mRNA could also initiate translation from the 5'-terminal AUG codon without the need for initiation factors'4. In fact, eukaryotic initiation is supported by quite a lot of different initiation factors, although complete reconstitution of the initiation process of wheat is in progress in Browning's group. Unfortunately, we have not obtained any successful result concerning IRES and leaderless inititiation. Therefore, we are now trying to purify eIF2, which may accelerate the leaderless initiation.
Time (min) Figure 4. Incorporation of radioactive Phe into polypeptide fraction during poly(U)-dependent poly(Phe) synthesis supported by the fractions. Poly(Phe) synthesis was performed with eEFIA and eEF2 (open squares), the s78 fraction and eEFIA (filled squares), or s78 only (filled triangles). As shown in the results section, we could confirm that eEFIA could be obtained in good amount (but with a moderate purity) by the method. The eEFIA fraction could at least be useful for the assay of the eEF2 activity in poly(Phe) synthesis experiments. In fact, the ribosome fraction used in this study contained very small amount of eEF2, which was not detected in gel electrophoresis analyses. So far, it is not easy to remove the eEF2 activity completely from the ribosome fraction. eEF2 did not give any sharp peak in the DE52 and P1i1
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