In Vitro Reconstitution of Yeast Translation System Capable of Synthesizing Long Polypeptide and Recapitulating Programmed Ribosome Stalling

The rates of translation elongation or termination in eukaryotes are modulated through cooperative molecular interactions involving mRNA, the ribosome, aminoacyl- and nascent polypeptidyl-tRNAs, and translation factors. To investigate the molecular mechanisms underlying these processes, we developed an in vitro translation system from yeast, reconstituted with purified translation elongation and termination factors, utilizing CrPV IGR IRES-containing mRNA, which functions in the absence of initiation factors. The system is capable of synthesizing not only short oligopeptides but also long reporter proteins such as nanoluciferase. By setting appropriate translation reaction conditions, such as the Mg2+/polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) is properly recapitulated in this system. We describe protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention critical parameters for setting up the translation reaction conditions. This reconstituted translation system not only facilitates biochemical analyses of translation but is also useful for various applications, such as structural and functional studies with the aim of designing drugs that act on eukaryotic ribosomes, and the development of systems for producing novel functional proteins by incorporating unnatural amino acids by eukaryotic ribosomes.


Introduction
The reconstitution of biological processes is critical for the investigation of the underlying molecular mechanisms. Regarding the translation of mRNA, some reconstituted translation systems have, so far, been developed with bacterial (Escherichia coli [1], Thermus thermophilus [2], Mycobacterium [3]), human [4], and mammalian mitochondrial factors [5]. A system from yeast Saccharomyces cerevisiae has been long-awaited, as yeast is one of the best-characterized model eukaryotes with a variety of resources. For yeast, the individual steps of translation have been partially reconstituted: initiation [6,7], peptide elongation [8][9][10][11], and termination and ribosome recycling [11][12][13][14]. Combination of these in vitro systems allowed synthesizing oligopeptides, and the direct measurement of the kinetics of translation elongation, by manipulating mRNA coding sequence, tRNA identity and concentration, as well as ribosome and translation factor composition [15]. However, until recently, the translation of a long peptide has not been attempted. This is partly because cap-dependent translation initiation and tRNA aminoacylation are complicated biological processes. Cap-dependent initiation is the most complex stage of translation in yeast, requiring at least 12 initiation factors, with many being composed of several peptides [6]. Moreover, the tRNA aminoacylation process is also highly complex, as 20 2 of 43 different aminoacyl-tRNA synthetases and their cofactors are involved in charging tRNAs with their cognate amino acids [16,17].
We have recently developed an in vitro reconstituted translation system that is capable of synthesizing long peptides [18]. In this system, in order to bypass the complex initiation process, we exploited CrPV IGR IRES (intergenic region internal ribosome entry site sequence from cricket paralysis virus)-containing mRNA, whereby yeast 80S ribosomes initiate in the absence of initiator tRNA or any eukaryotic translation initiation factors [19,20]. The translation elongation process was reconstituted by using yeast elongation factors (eEF1A, eEF2, and the fungal-specific elongation factor eEF3 [15,[21][22][23]) and pre-charged aminoacyl-tRNAs. To prepare aminoacyl-tRNAs, a yeast tRNA mixture was charged by yeast S100 extract, which we found to be capable of template-dependent synthesis of long polypeptides. Termination factors (eRF1 and eRF3) and recycling factors (Rli1, Hbs1, and Dom34) were further combined, in order to complete the reconstituted in vitro translation system. By setting appropriate translation reaction conditions, such as the Mg 2+ /polyamine concentration, the arrest of translation elongation by known ribosome-stalling sequences (e.g., polyproline and CGA codon repeats) can be properly recapitulated in this system. This system made it possible to analyze the translation elongation and termination, with greater control over the length and sequence of mRNA and nascent peptides. For example, by utilizing this system, we have examined the role of the eukaryotic translation factor eIF5A and its hypusine modification in translating the polyproline sequence within long open reading frames. We demonstrated that the requirement of the hypusine modification of eIF5A to alleviate the polyproline-mediated ribosome-stalling depends on the location and length of the polyproline motif in the protein [24].
Here, we describe detailed protocols for the preparation of the system components, manipulation of the system, and detection of the translation products. We also mention the translation reaction conditions, in order to recapitulate the programmed ribosome stalling. This reconstituted yeast translation system facilitates biochemical analyses of translation elongation, termination, and ribosome recycling on natural mRNAs, and also provides a framework for studying co-translational events, such as protein folding, targeting, and degradation through the ribosome-associated quality control (RQC) pathway. Moreover, the system is potentially useful for various applications; for example, it is suitable for use in structural and functional studies to design drugs that act on eukaryotic ribosomes, such as antibiotics against fungi. As the system allows for research into the effect of mRNA-or nascent peptide-context on translation termination, it could also be applied for the development of readthrough-inducing agents for treating genetic diseases caused by nonsense mutations. Other applications of this system include the template-dependent synthesis of proteins containing unnatural amino acids by eukaryotic ribosomes, using synthetic tRNAs that have been appropriately charged for example, with ribozymes. In combination with ribosome-or mRNA-display systems, this system could serve as a powerful in vitro screening system for novel functional proteins, such as antimicrobial peptides with cytotoxicity.

Experimental Design
This protocol describes the reconstitution of the yeast translation system in vitro, which is capable of synthesizing long polypeptides following CrPV-IRES-mediated translation initiation, as well as recapitulating the translation elongation arrest by known downregulating sequences, such as the polyproline sequence. As illustrated in Figure 1, the procedure includes five parts: (i) preparation of yeast translation factors (elongation factors, termination factors, recycling factors, ribosomes, tRNAs); (ii) preparation of aminoacyl-tRNAs by charging yeast crude tRNA mixtures using yeast S100 extract; (iii) preparation of CrPV IRES-containing mRNA by in vitro transcription using T7 RNA polymerase; (iv) the translation reaction; and (v) analysis of the translation products. In the following, we explain the parameters that critically determine the properties of the reconstituted translation system.

eEF1A concentration
A high eEF1A concentration-that is, 10-to 20-fold relative to the ribosome concentration-is required for efficient translation reactions, especially to synthesize long polypeptides. For example, the synthesis yield of the reporter protein nanoluciferase (nLuc) using an equivalent amount of eEF1A to ribosomes drops three orders of magnitude from that when using 10-fold eEF1A; that is, the yield of nLuc drops from 0.03 nM to 0.03 pM [18]. We found that such a high eEF1A concentration is important to ensure the dominance of IRES-mediated translation initiation over IRES-independent random internal translation initiation [18]. In the CrPV IRES-mediated translation initiation, the delivery of the first tRNA by eEF1A governs the overall efficiency of initiation [19,25]. Thus, for the long mRNA encoding long polypeptides, ternary complexes (eEF1A•aa-tRNA•GTP) may possibly be consumed by the IRES-independent random internal translation initiation, thereby inhibiting the IRES-dependent translation initiation. Indeed, a high eEF1A concentration is not strictly required when the short mRNA encoding an oligopeptide is translated with the system. The synthesis of oligopeptides using an equivalent amount of eEF1A to ribosomes is reduced to, at most, one-fifth of that using 10-fold eEF1A (i.e., the yield of oligopeptide synthesis drops from 3 nM to 0.6 nM [18]), whereby the required eEF1A amount is roughly proportional to the number of amino acids to be polymerized. Magnesium/polyamine concentration It is important to set an appropriate Mg 2+ and polyamine concentrations in the reconstituted yeast translation system, in order to recapitulate polyproline-mediated ribosome stalling. We have previously demonstrated that polyproline arrests translation in a manner similar to 'intrinsic ribosome destabilization (IRD)' [24]. IRD is a recently discovered phenomenon in bacteria, where consecutive and proline-intermitted acidic amino acids destabilize the 70S ribosome from within the peptide tunnel and abort translation [26]. Thus, in a yeast reconstituted translation system, under conditions with relatively lower Mg 2+ , polyproline destabilizes both the peptidylpolyproline-tRNA itself and the ribosome, inhibiting the peptidyl transfer reaction. However, under conditions with relatively higher Mg 2+ (where ribosomes are stable), the disorder of the peptidylpolyproline-tRNA is resolved, consequently alleviating polyproline-mediated ribosome stalling. To analyze polyproline-mediated ribosome stalling or other potential magnesium/polyaminedependent translation regulation, we usually set the concentration of Mg 2+ in a range of about 5 to 7 mM, in the presence of 0.25 mM spermidine. The precise Mg 2+ concentration needs to be determined, depending on the lot of 80S ribosome preparation, as described later (see Figure 2). Ribosome stalling is alleviated and no longer observed at Mg 2+ concentrations above a specific concentration between 5 and 7 mM.
We commonly use mRNA and pre-charged aa-tRNAs for the translation reaction in the reconstituted translation system ( Figure 1). On the other hand, by including a DNA template, T7 RNA polymerase, and/or purified aminoacyl-tRNA synthetases (human aminoacyl-tRNA synthetases [4]) in the system, it is also possible to perform a translation reaction coupled with transcription and/or aminoacylation reaction, which greatly simplifies the experiments and saves time (see Section 3.4); however, care must be taken that the T7 RNA polymerase and aminoacyl-tRNA synthetases consume triphosphate nucleotides which chelate Mg 2+ during the reaction, such that the control of Mg 2+ concentration is difficult in such transcription-and aminoacylation-coupled systems.

Cell Culture
LB medium: Mix 10 g of bacto tryptone, 5 g of bacto yeast extract, and 10 g of NaCl, and then, add water up to 1 L. Thereafter, autoclave it. 2xYT medium: Mix 16 g of bacto tryptone, 10 g of bacto yeast extract, and 5 g of NaCl, and then, add water up to 1 L. Thereafter, autoclave it. YPD medium: Mix 9 g of bacto yeast extract, 18 g of hipolypepton, and 18 g of D(+)glucose, and then, add water up to 900 mL. Thereafter, autoclave it. Bacto peptone (Thermo Fisher Scientific) can be used instead of hipolypepton (Wako).

SC-Ura medium containing 1% (wt/vol) raffinose:
To prepare 400 mL of medium, mix 2.67 g of difco yeast nitrogen base without amino acids and 0.77 g of dropout mix without Ura, and then, add water up to 350 mL. Add 1 M NaOH (approximately 4 mL) into the medium, until the pH reaches 7-8. After pH adjustment, add water up to 360 mL into the medium. Thereafter, autoclave it. Cool the medium to room temperature and add 40 mL of 10% (wt/vol) raffinose. To prepare 720 mL of medium, mix 5.33 g of difco yeast nitrogen base without amino acids and 1.54 g of dropout mix without Ura, and then, add water up to 600 mL. Add 1 M NaOH (approximately 7 mL) into the medium, until the pH reaches 7-8. After pH adjustment, add water up to 640 mL into the medium. Thereafter, autoclave it. Cool the medium to room temperature and add 80 mL of 10% (wt/vol) raffinose.

Preparation of Yeast Translation Factors
All translation factors are checked for purity by SDS-PAGE analysis after purification (see [18] for the typical results). The concentrations of purified translation factors are determined by the Bradford Assay or by measuring the absorption spectrum and using the absorption coefficient (Table S1). The yields of the factors are summarized in Table S2. Verify the activities of the purified factors, according to Section 3.4.3, for the translation reaction method, and refer to Table S4 for the appropriate translation reaction condition for each translation factor.

eEF1A
Cell culture

1.
From a glycerol stock, streak a sample of YPH499 yeast cells on a YPD plate and grow at 30 • C for 2 days.

2.
Collect the cells from the plate using a disposable loop, inoculate a 150 mL culture of YPD medium in a 500 mL flask with the cells, and then, grow overnight (approximately 16 h) at 30 • C with vigorous shaking at 170 rpm.

3.
Inoculate six 900 mL culture of YPD medium in 2 L baffled flasks with 20 mL of the overnight culture per flask and grow at 30 • C with vigorous shaking at 170 rpm until the culture reaches an OD 600 of 4.0. This should take approximately 9 h. 4.
Harvest the cells by centrifugation at 9000× g for 10 min at 4 • C.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 4.0 g/L culture. 7.
Resuspend with an equal amount of lysis buffer (1 mL buffer per 1 g cells) and dispense the cells, through micropipettes, into liquid nitrogen to make frozen yeast droplets. PAUSE STEP Frozen cells can be stored at −80 • C for approximately one month.

1.
Disrupt the cells by mechanical grinding (see Appendix A).

2.
Thaw the frozen powder of ground cells at 4 • C and centrifuge at 7500× g for 10 min at 4 • C. Thereafter, transfer the supernatant to a clean tube (Sup 1, approximately 20 mL).

3.
Resuspend the resulting pellets with 30 mL of lysis buffer and centrifuge at 7500× g for 10 min at 4 • C. Thereafter, transfer the supernatant to a clean tube (Sup 2, approximately 30 mL).
NOTE: Take care to minimize contamination from either the lipid fraction at the very top or the cell debris at the bottom of the tube.

1.
Incubate S100 and 15 mL of Q sepharose Fast Flow resin (Cytiva) pre-equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Sediment the resin by centrifugation at 500× g for 10 min, then transfer the supernatant to a clean tube (flow-through fraction, approximately 40 mL). 3.
Add 1 column volume of lysis buffer to the resin and gently mix the slurry. Sediment the resin by centrifugation at 500× g for 10 min, and then, transfer the supernatant to a clean tube (wash fraction, approximately 15 mL).
Methods Protoc. 2021, 4, x FOR PEER REVIEW 17 of 43 6. Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 °C. The pellet weight should be 4.0 g/L culture. 7. Resuspend with an equal amount of lysis buffer (1 mL buffer per 1 g cells) and dispense the cells, through micropipettes, into liquid nitrogen to make frozen yeast droplets. 5. Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF1A. Typically, eEF1A is eluted at about 65% Butyl B buffer as a major peak, and the volume of the pool is approximately 30 mL. Be careful not to collect the base of the peak, which contain impurities. 6. Dialyze the sample against 3 L of eEF1A stock buffer at 4 °C overnight with two changes of buffer; dialyze 2 h, 16 h, and another 2 h.

PAUSE STEP
CRITICAL STEP Residual ammonium sulfate in the purified eEF1A inhibits the translation.
7. Concentrate the purified protein to approximately 5 mg/mL using Amicon Ultra Centrifugal Filters (10 kDa cut-off) (MERCK). 8. Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF1A ( Figure S1). 9. The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 °C.
The yield is about 1 mg/L culture (Table S2).

1.
To check the activity of the purified eEF1A, it is recommended to perform the translation reaction both in the presence and absence of eEF3, in order to analyze the eEF3 contamination levels. The purified eEF1A is contaminated with a small amount of eEF3 (<1%), as estimated by western blotting analysis. Note here that a high concentration of eEF1A (10-20 times the ribosomal concentration) is utilized in a standard translation reaction condition. Therefore, only when extremely pure eEF1A is obtained, the omission of eEF3 reduces the translation efficiency by about half. Otherwise, there is little effect of eEF3 on the translation efficiency. In our hands, further purification by the gel filtration do not eliminate the contaminating eEF3 from the purified eEF1A.

eEF2
Cell culture 1. From a glycerol stock, streak a sample of TKY675/p416GPD-eEF2 yeast cells on a YPD plate and grow at 30 °C for 2 days. CRITICAL STEP Residual ammonium sulfate in the purified eEF1A inhibits the translation.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF1A ( Figure S1). 9.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.
The yield is about 1 mg/L culture (Table S2).

1.
To check the activity of the purified eEF1A, it is recommended to perform the translation reaction both in the presence and absence of eEF3, in order to analyze the eEF3 contamination levels. The purified eEF1A is contaminated with a small amount of eEF3 (<1%), as estimated by western blotting analysis. Note here that a high concentration of eEF1A (10-20 times the ribosomal concentration) is utilized in a standard translation reaction condition. Therefore, only when extremely pure eEF1A is obtained, the omission of eEF3 reduces the translation efficiency by about half. Otherwise, there is little effect of eEF3 on the translation efficiency. In our hands, further purification by the gel filtration do not eliminate the contaminating eEF3 from the purified eEF1A.

eEF2
Cell culture

1.
From a glycerol stock, streak a sample of TKY675/p416GPD-eEF2 yeast cells on a YPD plate and grow at 30 • C for 2 days.

2.
Collect the cells from the plate using a disposable loop, inoculate a 300 mL culture of YPD medium in a 500 mL flask with the cells, and then grow overnight (approximately 16 h) at 30 • C with vigorous shaking at 170 rpm.

3.
Inoculate twelve 900 mL culture of YPD medium in 2 L baffled flasks with 20 mL of the overnight culture per flask and grow at 30 • C with vigorous shaking at 170 rpm, until the culture reaches an OD 600 of 4.0. This should take approximately 24 h.

4.
Harvest the cells by centrifugation at 9000× g for 10 min at 4 • C.
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 4.8 g/L culture. Stir until the mixture is homogenous, then stir for 20 min at room temperature.

3.
Centrifuge the samples at 7500× g for 10 min at 4 • C and keep the supernatants.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 2 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with Ni-NTA wash Buffer I on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 2 columns). Collect the flow-through fractions in a clean tube.

3.
Wash with 20 column volumes of Ni-NTA wash Buffer I (20 mL buffer/column). Collect the wash fractions into clean tubes.

4.
Wash with 20 column volumes of Ni-NTA wash Buffer II (20 mL buffer/column). Collect the wash fractions into clean tubes.

5.
Wash with 30 column volumes of Ni-NTA wash Buffer I (30 mL buffer/column). Collect the wash fractions into clean tubes. 6.
Analyze the fractions by 10% SDS-PAGE and pool fractions containing eEF2. 8.
Dialyze the sample against 1 L of dialysis buffer at 4 • C overnight with a change of buffer after 1 h. 9.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.
Purification by HiTrap Q column chromatography

1.
Load the sample at a flow rate of 1.0 mL/min onto a Hitrap Q HP column (5 mL, Cytiva) which has been equilibrated with 10% (100 mM KCl) B buffer.

2.
After washing with 5 column volumes of 10% (100 mM KCl) Q B buffer, elute with a linear gradient from 10 to 25% (100 to 250 mM KCl) Q B buffer in 15 column volumes at a flow rate of 2.0 mL/min, collecting 1.0 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF2. Typically, eEF2 is eluted at about 23% B buffer as a major peak, and the volume of the pool is approximately 8 mL. Be careful not to collect the base of the peak, which contains impurities.

4.
Dialyze the sample against 1 L of eEF2 stock buffer at 4 • C overnight, with a change of buffer after 1 h.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF2 ( Figure S1). 7.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.

eEF3
Cell culture
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 2.0 g/L culture.

Disrupt the cells by sonication (see Appendix A). 2.
Centrifuge the disrupted cells at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.

3.
Centrifuge the sample again at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 2 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 2 columns). Collect the flow-through fractions in clean tubes.

3.
Wash with 100 column volumes of Ni-NTA wash buffer (100 mL buffer/column). Collect the wash fractions and transfer to clean tubes.

4.
Elute with 5 column volumes of Ni-NTA elution buffer (5 mL buffer/column). Collect the eluate fractions and transfer to clean tubes.
Dialyze the sample against 1 L of dialysis buffer at 4 • C overnight with a change of buffer after 1 h. 7.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.

1.
After diluting the sample 2-fold by adding an equal volume of 0% (0 mM KCl) Q B buffer, load the sample at a flow rate of 0.5 mL/min onto a HiTrap Q column (5 mL, Cytiva) which has been equilibrated with 5% (50 mM KCl) B buffer.

2.
After washing with 5 column volumes of 5% (50 mM KCl) Q B buffer, elute with a linear gradient from 5 to 35% (50 to 350 mM KCl) Q B buffer in 15 column volumes at a flow rate of 0.5 mL/min, collecting 1 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF3. Typically, eEF3 is eluted at about 17% Q B buffer as a major peak, and the volume of the pool is approximately 7 mL. Be careful not to collect the base of the peak, which contains impurities.

4.
Dialyze the sample against 1 L of eEF3 stock buffer at 4 • C overnight, with a change of buffer after 1 h.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF3 ( Figure S1). 7.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.

eRF1
Cell culture
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 8.0 g/L culture.

1.
Disrupt the cells by sonication (see Appendix A).

2.
Centrifuge the disrupted cells at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.

3.
Centrifuge the sample again at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 2 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 2 columns). Collect the flow-through fractions in clean tubes.

3.
Wash with 100 column volumes of Ni-NTA wash buffer (100 mL buffer/column). Collect the wash fractions in clean tubes.
Dialyze the sample against 1 L of stock buffer at 4 • C overnight, with a change of buffer after 1 h.

7.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.

Purification by HiTrap Q column chromatography
1.
Load the sample at a flow rate of 1.0 mL/min onto a HiTrap Q HP column (20 mL, Cytiva) that has been equilibrated with 10% (100 mM KCl) B buffer.

2.
Wash with 5 column volumes of 20% (200 mM KCl) Q B buffer, then with another 5 column volumes of 30% (300 mM KCl) Q B buffer, at a flow rate of 2.0 mL/min.

3.
Elute with a linear gradient from 30 to 60% (300 to 600 mM KCl) B buffer in 15 column volumes at a flow rate of 2.0 mL/min, collecting 4 mL fractions.

4.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eRF1. Typically, eRF1 is eluted at about 36% B buffer as a major peak, and the volume of the pool is approximately 20 mL. Be careful not to collect the base of the peak, which contains impurities.

5.
Dialyze the sample against 1 L of eRF1 stock buffer at 4 • C overnight, with a change of buffer after 1 h. 6.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eRF1 ( Figure S1). 8.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.
The yield is about 8.5 mg/L culture (Table S2).
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 7.8 g/L culture.

1.
Disrupt the cells by sonication (see Appendix A).

2.
Centrifuge the disrupted cells at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.

3.
Centrifuge the sample again at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 4 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 4 columns). Collect the flow-through fractions in clean tubes.

3.
Wash with 100 column volumes of Ni-NTA wash buffer (100 mL buffer/column). Collect the wash fractions in clean tubes.
Dialyze the sample against 1 L of stock buffer at 4 • C overnight, with a change of buffer after 1 h. 7.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.

Purification by HiTrap Q column chromatography
1.
Load the sample at a flow rate of 1.0 mL/min onto a HiTrap Q HP column (20 mL, Cytiva) which has been equilibrated with 10% (100 mM KCl) Q B buffer.

2.
After washing with 5 column volumes of 10% (100 mM KCl) Q B buffer, elute with a linear gradient from 10 to 30% (100 to 300 mM KCl) Q B buffer in 15 column volumes at a flow rate of 2.0 mL/min, collecting 4 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eRF3∆165. Typically, eRF3∆165 is eluted at about 17% B buffer as a major peak, and the volume of the pool is approximately 35 mL. Be careful not to collect the base of the peak, which contains impurities.

4.
Dialyze the sample against 2 L of eRF3∆165 stock buffer at 4 • C overnight, with a change of buffer after 1 h.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eRF3 ( Figure S1). 7.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.
The yield is about 24 mg/L culture (Table S2).

1.
eRF3∆165 is the more stable version of the full-length eRF3 with the deletion of the N-terminal amino acids not involved in function.

Dom34
Cell culture

2.
Pick a single colony and inoculate it into 30 mL of LB supplemented with 25 µg/mL kanamycin, 25 µg/mL chloramphenicol, and 1% (wt/vol) glucose, then grow overnight at 37 • C with vigorous shaking at 170 rpm.

3.
Inoculate two 1 L cultures of 2 × YT supplemented with 25 µg/mL kanamycin and 25 µg/mL chloramphenicol with 10 mL of the overnight culture per flask, and then, grow at 37 • C with vigorous shaking at 170 rpm, until the culture reaches an OD 600 of 0.5. This should take approximately 2 h. 4.
Induce protein expression by adding 1 mL of 100 mM IPTG (final 0.1 mM) per flask and grow overnight (approximately 12-16 h) at 18 • C with vigorous shaking at 170 rpm.

5.
Harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 6.
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C.

7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 9.6 g/L culture.
Methods Protoc. 2021, 4, x FOR PEER REVIEW 17 of 43 6. Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 °C. The pellet weight should be 4.0 g/L culture. 7. Resuspend with an equal amount of lysis buffer (1 mL buffer per 1 g cells) and dispense the cells, through micropipettes, into liquid nitrogen to make frozen yeast droplets.

Cell lysis
1. Disrupt the cells by mechanical grinding (see Appendix A). 2. Thaw the frozen powder of ground cells at 4 °C and centrifuge at 7500× g for 10 min at 4 °C. Thereafter, transfer the supernatant to a clean tube (Sup 1, approximately 20 mL). 3. Resuspend the resulting pellets with 30 mL of lysis buffer and centrifuge at 7500× g for 10 min at 4 °C. Thereafter, transfer the supernatant to a clean tube (Sup 2, approximately 30 mL). 4. Combine Sup 1 and Sup 2 and transfer to a clean ultracentrifuge tube. Centrifuge at 150,000× g (450,000 rpm, Type 70Ti, BECKMAN COULTER) for 3 h at 4 °C. Thereafter, transfer the supernatants to a clean tube (S100, approximately 40 mL). NOTE: Take care to minimize contamination from either the lipid fraction at the very top or the cell debris at the bottom of the tube.
Batch purification with Q sepharose 1. Incubate S100 and 15 mL of Q sepharose Fast Flow resin (Cytiva) pre-equilibrated with lysis buffer on a rotator at 4 °C for approximately 30 min. 2. Sediment the resin by centrifugation at 500× g for 10 min, then transfer the supernatant to a clean tube (flow-through fraction, approximately 40 mL). 3. Add 1 column volume of lysis buffer to the resin and gently mix the slurry. Sediment the resin by centrifugation at 500× g for 10 min, and then, transfer the supernatant to a clean tube (wash fraction, approximately 15 mL). 4. Combine the flow-through and wash fractions (approximately 55 mL).
PAUSE STEP Freeze the sample with liquid nitrogen; then, it can be stored at −80 °C for approximately 1 week.

1.
Disrupt the cells by sonication (see Appendix A).

2.
Centrifuge the disrupted cells at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.

3.
Centrifuge the sample again at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 4 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 4 columns). Collect the flow-through fractions in clean tubes.

3.
Wash with 100 column volumes of Ni-NTA wash buffer (100 mL buffer/column). Collect the wash fractions in clean tubes.
Dialyze the sample against 1 L of stock buffer at 4 • C overnight, with a change of buffer after 1 h. 7.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.

1.
After diluting the sample 2-fold by adding an equal volume of 0% (0 mM KCl) Q B buffer, load the sample at a flow rate of 2.0 mL/min onto a HiPrep Q HP column (20 mL, Cytiva) which has been equilibrated with 10% (100 mM KCl) Q B buffer.

2.
After washing with 5 column volumes of 10% (100 mM KCl) Q B buffer, elute with a linear gradient from 10 to 50% (100 to 500 mM KCl) Q B buffer in 15 column volumes at a flow rate of 2.0 mL/min, collecting 4 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain Dom34. Typically, Dom34 is eluted as a major peak at about 24% Q B buffer, with the former half of the peak containing impurities of about 38 kDa and the latter half of the peak containing impurities of about 40 kDa. Pool the latter half of the peak, which is approximately 50 mL.

4.
Dialyze the sample against 2 L of Dom34 stock buffer at 4 • C overnight with a change of buffer after 1 h.

2.
Load the sample at a flow rate of 0.5 mL/min onto a HiLoad 16/600 Superdex 200 pg column (Cytiva) which has been equilibrated with Dom34 stock buffer, collecting 1.2 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain Dom34. Typically, Dom34 is eluted at around 70 mL as a major peak, and the volume of the pool is approximately 7 mL. Be careful not to collect the base of the peak, which contains impurities.

5.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of Dom34 ( Figure S1).

6.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C. The yield is about 2.2 mg/L culture (Table S2).

Hbs1
Cell culture

2.
Pick a single colony and inoculate it into 30 mL of LB supplemented with 100 µg/mL ampicillin, and then, grow overnight at 37 • C with vigorous shaking at 170 rpm.

3.
Inoculate two 1 L cultures of 2 × YT supplemented with 100 µg/mL ampicillin with 10 mL of the overnight culture per flask, and then grow at 37 • C with vigorous shaking at 170 rpm, until the culture reaches an OD 600 of 0.5. This should take approximately 2 h. 4.
Induce protein expression by adding 1 mL of 100 mM IPTG (final 0.1 mM) per flask, and then, grow overnight (approximately 12-16 h) at 18 • C with vigorous shaking at 170 rpm.

5.
Harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 6.
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 7.8 g/L culture.

1.
Disrupt the cells by sonication (see Appendix A).

2.
Centrifuge the disrupted cells at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.

3.
Centrifuge the sample again at 10,000× g for 30 min at 4 • C. Thereafter, transfer the supernatants to a clean tube.
Purification with Ni-NTA agarose

1.
Incubate the lysate and 2 mL of Ni-NTA agarose resin (QIAGEN) equilibrated with lysis buffer on a rotator at 4 • C for approximately 30 min.

2.
Load the slurry into empty columns (1 mL resin/column, 2 columns). Collect the flow-through fractions in clean tubes.

3.
Wash with 100 column volumes of Ni-NTA wash buffer (100 mL buffer/column). Collect the wash fractions in clean tubes.
Dialyze the sample against 1 L of stock buffer at 4 • C overnight, with a change of buffer after 1 h. 7.
OPTIONAL STEP: Clarify the sample by centrifugation at 10,000× g for 15 min at 4 • C.

Purification by HiTrap Q column chromatography
1.
Load the sample at a flow rate of 1 mL/min onto a HiTrap Q column (10 mL, Cytiva) which has been equilibrated with 10% (100 mM KCl) Q B buffer.

2.
After washing with 3 column volumes of 10% (100 mM KCl) Q B buffer and then with another 3 column volumes of 25% (250 mM KCl) Q B buffer, elute with a linear gradient from 25 to 60% (250 to 600 mM KCl) Q B buffer in 15 column volumes at a flow rate of 2 mL/min, collecting 2 mL fractions.

3.
Analyze the fractions by 10% SDS-PAGE and pool fractions that contain Hbs1. Typically, Hbs1 is eluted at about 37% Q B buffer as a major peak, and the volume of the pool is approximately 10 mL. Be careful not to collect the base of the peak, which contains impurities.

4.
Dialyze the sample against 1 L of Hbs1 stock buffer at 4 • C overnight, with a change of buffer after 1 h. 5.
Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of Hbs1 ( Figure S1). 7.
The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C.
The yield is about 1.6 mg/L culture (Table S2).

Rli1
Cell culture Harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 6.
Resuspend with 1 L of 1% (wt/vol) KCl and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. 7.
Resuspend with 1 L of lysis buffer and harvest the cells by centrifugation at 9000× g for 10 min at 4 • C. The pellet weight should be 4.0 g/L culture. 8.
Resuspend with an equal amount of lysis buffer (1 mL buffer per 1 g cells) and dispense the cells through micropipettes into liquid nitrogen, to form frozen yeast droplets.  5. Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF1A. Typically, eEF1A is eluted at about 65% Butyl B buffer as a major peak, and the volume of the pool is approximately 30 mL. Be careful not to collect the base of the peak, which contain impurities. 6. Dialyze the sample against 3 L of eEF1A stock buffer at 4 °C overnight with two changes of buffer; dialyze 2 h, 16 h, and another 2 h.
CRITICAL STEP Residual ammonium sulfate in the purified eEF1A inhibits the translation.
7. Concentrate the purified protein to approximately 5 mg/mL using Amicon Ultra Centrifugal Filters (10 kDa cut-off) (MERCK). 8. Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF1A ( Figure S1). 9. The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 °C.
The yield is about 1 mg/L culture (Table S2).

1.
To check the activity of the purified eEF1A, it is recommended to perform the translation reaction both in the presence and absence of eEF3, in order to analyze the eEF3 contamination levels. The purified eEF1A is contaminated with a small amount of eEF3 (<1%), as estimated by western blotting analysis. Note here that a high concentration of eEF1A (10-20 times the ribosomal concentration) is utilized in a standard translation reaction condition. Therefore, only when extremely pure eEF1A is obtained, the omission of eEF3 reduces the translation efficiency by about half. Otherwise, there is little effect of eEF3 on the translation efficiency. In our hands, further purification by the gel filtration do not eliminate the contaminating eEF3 from the purified eEF1A.

eEF2
Cell culture CRITICAL STEP: be careful not to pool the fractions of 60S subunits, which appear as the shoulder at the beginning of the major peak and whose A 260 are usually < 2.

9.
Transfer the 80S pool into a 45Ti polycarbonate ultracentrifuge tube (BECKMAN COULTER). Add 50% B buffer, such that the sample volume becomes 50 mL. If the volume of the 80S pool is greater than 50 mL, divide the sample into two 45Ti polycarbonate ultracentrifuge tubes and add 50% B buffer, such that the sample volume of each tube is 50 mL. 10. Place 20 mL of sucrose cushion Buffer II at the very bottom of the 45Ti tube(s) using a syringe. 11. Centrifuge at 100,000× g (36,000 rpm, Type 45Ti, BECKMAN COULTER) for 16 h at 4 • C. Thereafter, aspirate off the supernatants. 12. Add approximately 50 µL of 5/100 buffer per tube and dissolve the pellets by gently shaking for approximately 1 h at 4 • C, and then, transfer to clean tubes. Rinse the tube with a small amount of 5/100 buffer (approximately 10 µL) and combine it with the recovered ribosomes. 13. Measure the A 260 of the sample using a spectrometer and determine the concentration of 80S ribosomes, estimating that the 80S ribosomes at 1.0 A 260 correspond to 20 pmol. 14. The purified 80S ribosomes are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 • C. The yield is about 110 pmol/L culture (Table S2).

Analysis of ribosomal proteins
1. Add 2.5 µL of 5× LiDS dye to 10 pmol of the purified 80S ribosomes in 10 µL of 5/100 buffer and mix well.

2.
Apply the sample to a 15% SDS-PAGE gel, and then, visualize the protein bands by CBB R-250 staining.

4.
After the incubation at room temperature for 5 min, add 200 µL of chloroform to the sample, and then, vortex well.

5.
After the incubation at room temperature for 5 min, centrifuge at 16,000× g for 10 min at 27 • C. Thereafter, remove the aqueous (upper) phase (approximately 400 µL) and transfer to a clean tube. 6.
Add 40 µL of 3M KOAc (pH 5.5) and 500 µL of ice-cold isopropanol to the sample and vortex well. 7.
Centrifuge at 16,000× g for 30 min at 4 • C. Thereafter, aspirate off the supernatant and save the pellet. 8.
Centrifuge at 16,000× g for 5 min at 4 • C. Thereafter, aspirate off the supernatant and save the pellet.
5. Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF1A. Typically, eEF1A is eluted at about 65% Butyl B buffer as a major peak, and the volume of the pool is approximately 30 mL. Be careful not to collect the base of the peak, which contain impurities. 6. Dialyze the sample against 3 L of eEF1A stock buffer at 4 °C overnight with two changes of buffer; dialyze 2 h, 16 h, and another 2 h.
CRITICAL STEP Residual ammonium sulfate in the purified eEF1A inhibits the translation.
7. Concentrate the purified protein to approximately 5 mg/mL using Amicon Ultra Centrifugal Filters (10 kDa cut-off) (MERCK). 8. Determine the concentration of the protein by the Bradford Assay or by using the adsorption coefficient of eEF1A ( Figure S1). 9. The purified factors are aliquoted, fast-frozen in liquid nitrogen, and stored at −80 °C.
The yield is about 1 mg/L culture (Table S2).

1.
To check the activity of the purified eEF1A, it is recommended to perform the translation reaction both in the presence and absence of eEF3, in order to analyze the eEF3 contamination levels. The purified eEF1A is contaminated with a small amount of eEF3 (<1%), as estimated by western blotting analysis. Note here that a high concentration of eEF1A (10-20 times the ribosomal concentration) is utilized in a standard translation reaction condition. Therefore, only when extremely pure eEF1A is ob-CRITICAL STEP: it is recommended that the columns be set in a cold room.
11. Load 150 µL of sample per column and allow the sample to enter the gel bed completely. 12. Load 350 µL of ice-cold equilibration buffer per column, such that combined volume of the sample and equilibration buffer equals 500 µL. Allow the equilibration buffer to enter the gel bed completely. 13. Elute with 560 µL of ice-cold equilibration buffer per column, collecting the sample in a 5 mL tube.
14. Add 2 volumes (1.12 mL) of ice-cold 100% ethanol to the eluate per tube and vortex well. 15. Centrifuge the sample at maximum speed (~15,000× g) for 30 min at 4 • C. Thereafter, aspirate off the supernatant. 16. Gently add 2 mL of ice-cold 70% (vol/vol) ethanol per tube, to rinse the pellet. 17. Centrifuge the sample at maximum speed (~15,000× g) for 5 min at 4 • C. Thereafter, aspirate off the supernatant. Remove as much ethanol as possible. 18. Dry the pellet for 5 min at 27 • C. Do not over-dry the pellets; they should be slightly wet. 19. Resuspend the pellet with 100 µL of ice-cold 50 mM KOAc (pH 5.5). 20. The aminoacyl-tRNAs are aliquoted in approximately single-use aliquots (60 µL), fast-frozen in liquid nitrogen, and stored at −80 • C, where they usually remain stable for months. Vigorously shake the samples for 30 min at 27 • C. 9.
Measure the radioactivity of the samples using a scintillation counter.

1.
Approximately 8 pmol of [ 35 S]methionine is usually charged per one A 260 unit of tRNA mixture.

2.
If the aminoacylation efficiency is inefficient, the volume of S100 in the reaction can be increased up to 1/5 reaction volumes (200 µL).

3.
Verify the translation ability of the aminoacyl-tRNAs, referring to Section 3.4.3 for the translation reaction method, and to Table S4 for the translation reaction condition.

Preparation of CrPV-IRES Containing mRNA
Prepare the desired mRNAs by in vitro transcription using T7 RNA polymerase and the template DNAs, according to the standard protocols (for details, see Appendix B). The template DNA sequences for the mRNAs used in Figure 2 are shown in Table S6.

Transcription-Aminoacylation-Translation Coupled Reaction
The protocols in this section describe a standard reaction, where a DNA template and T7 RNA polymerase are included for the mRNA transcription, and human aminoacyl-tRNA synthetase mixture [4] is included for aminoacylation. The Mg 2+ and polyamine concentration in the reaction, [Mg/SPD/SP], is set as [9/2/0.1], in order to sustain the efficient transcription and aminoacylation reactions. Note that polyproline-mediated translation arrest is not recapitulated under this condition.

1.
Prepare the "buffer mixture," "enzyme mixture," and "DNA solution" (total 20 µL) on ice, as follows ( Table 2):   RNase-free water (to 20 µL)  RNase-free water (to 20 µL) * 1 The components listed in Table S5 are dissolved in stock buffer containing the indicated Mg 2+ concentrations [1,4,18]. X is determined by taking into account the concentration of Mg 2+ derived from the components, such that the final concentration of Mg 2+ in the reaction is 9 mM. * 2 When analyzing the [ 35 S]methionine-labelled translation products, add 1 µL of [ 35 S]methionine (PerkinElmer, NEG009A) instead of cold methionine.

Translation Reaction without Coupled Reaction
The protocols in this section describe a standard reaction where the translation reaction is separated from transcription and aminoacylation reactions; mRNAs are utilized instead of DNA template and T7 RNA polymerase, and a pre-charged aminoacyl-tRNA mixture is used instead of a human aminoacyl-tRNA synthetase mixture [4]. The Mg 2+ and polyamine concentration in the reaction, [Mg/SPD/SP], is usually set as [5-7/0.25/0], such that polyproline-mediated translation arrest is properly recapitulated. The precise Mg 2+ concentration needs to be determined, depending on the lot of 80S ribosome preparation (see Figure 2).

1.
Prepare the "buffer mixture," "enzyme mixture," "aminoacyl-tRNA mixture," and "mRNA solution" (total 20 µL) on ice, as follows (Table 4):  RNase-free water (to 20 µL) Aminoacyl-tRNA mixture Aminoacyl-tRNA (0.125 A 260 units) and 7 mM, and the nanoluciferase activities were measured ( Figure 2B). With the ribosomes of this preparation, we consider that the appropriate condition for further studies is 6 mM Mg 2+ , for the following reasons: (i) polyproline-mediated translation arrest was properly observed ( Figure 2B, Panel 5), and (ii) the translation efficiency of the no-motif mRNA in the absence and presence of eIF5A was approximately the same at a reaction time of 120 min ( Figure 2B, Panel 2). Note that, under the 7 mM Mg 2+ condition, polyproline-mediated translation arrest was no longer observed ( Figure 2B, Panel 6). Moreover, translation of no-motif mRNA was considerably inhibited by eIF5A ( Figure 2B, Panel 3). The reason for this is still unclear; one possibility is that eIF5A promotes premature termination at several sites in the ORF (for a detailed discussion, see [24]). On the other hand, under the 5 mM Mg 2+ condition, the translation initiation and the early elongation process were presumably inefficient in the absence of eIF5A ( Figure 2B, Panel 1). Consequently, the effect of eIF5A on the translation of the polyproline sequence, per se, would not be appropriately evaluated under such a condition ( Figure 2B, Panel 4). Figure 2C shows the analysis of the translation products at 6 mM Mg 2+ condition by Tricine SDS-PAGE. In agreement with the nanoluciferase activities, the truncated proteins are observed from the Prox4 mRNAs, and the addition of eIF5A stimulates the production of full-length nanoLuciferase.
For other examples of detailed analyses of translation elongation regulation with the reconstituted yeast translation system, see [24]. Ribosome stalling by the polyproline sequence in either oligopeptides or long polypeptides, CGA codon repeats, and the polytryptophan sequence have been analyzed using the methods described herein. The effect of Mg 2+ /polyamine on the translation of the polyproline sequence has also been presented in detail.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/mps4030045/s1, Table S1. Summary of the experimentally estimated absorption coefficients of the purified factors, Table S2. Summary of the expression and purification of the components for the reconstituted translation system, Table S3. Yeast strains used for the purification of the components for the reconstituted translation system, Table S4. Translation reaction condition for checking the activity of the components in the reconstituted translation system, Table S5. Components in the reconstituted translation system dissolved in buffer that contained Mg 2+ , Table S6. Sequence of template DNA.

Acknowledgments:
The authors sincerely thank Akira Kaji and Hideko Kaji for the TKY675 strain and the plasmid containing the CrPV IGR IRES cDNA, Hiroaki Imataka for human aminoacyl-tRNA synthetases, and Takuya Ueda and all the members of the laboratory for their helpful discussions and continuous support.

Conflicts of Interest:
The authors declare no conflict of interest.

1.
Fill the grinder (Retsch, Mortar Grinder RM200) with liquid nitrogen and preliminarily run the mortar before adding frozen cells.
5. Analyze the fractions by 10% SDS-PAGE and pool fractions that contain eEF1A. Typically, eEF1A is eluted at about 65% Butyl B buffer as a major peak, and the volume of the pool is approximately 30 mL. Be careful not to collect the base of the peak, which contain impurities. 6. Dialyze the sample against 3 L of eEF1A stock buffer at 4 °C overnight with two changes of buffer; dialyze 2 h, 16 h, and another 2 h.