Methods 36 (2005) 299–304

www.elsevier.com/locate/ymeth

Protein synthesis by pure translation systems

Yoshihiro Shimizu a, Takashi Kanamori b, Takuya Ueda a,¤

a Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bldg. FSB-4015-1-5 Kashiwanoha,Kashiwa, Chiba Prefecture 277-8562, Japan

b Post Genome Institute Co., Ltd., 6F Hongo Ishiwata Bldg., 3-38-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Accepted 28 April 2005

Abstract

We have developed a partially recombinant, cell-free, protein-synthesis system reconstituted solely from those essential elementsof the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE). It provides higher reactioncontrollability in comparison to crude cell-free protein-synthesis systems for translation studies and biotechnology applications. ThePURE system stands out among translation methods in that it provides not only a simple and unique “reverse” puriWcation methodof separating the synthesized protein from reaction mixture, but also that the system can be tailor-made according to individual pro-tein requirements. In this paper, two new approaches to obtaining active proteins are described: the use of molecular chaperones, andmodiWcation of the reaction conditions. Several possible applications of the PURE system are also discussed. 2005 Elsevier Inc. All rights reserved.

Keywords: Cell-free protein synthesis; Translation; Protein folding; Molecular chaperone; DisulWde bond formation; Unnatural amino acid; Proteinselection

1. Introduction

Research is moving from the gathering of genomicsequences to protein expression for the analysis ofsequence function and utility. In accordance with thissequence-to-function paradigm shift, the preparation oflarge quantities of numerous active proteins using a sim-ple yet sophisticated approach is expected in many Weldssuch as chemical biology, structural biology, and phar-maceutical biology.

To prepare large amounts of these proteins, in vivo pro-duction by a recombinant DNA system is a very powerfultool owing to its productivity. Protein expression in 100mlculture of E. coli cells recovers milligram of puriWed pro-tein. Fusion protein strategies such as the hexa-histidinetag (His-tag) [1] and GST fusion protein [2] have simpliWedthe puriWcation procedures so that protein can be puriWed

* Corresponding author. Fax: +81 0 471 36 3642.E-mail address: ueda@k.u-tokyo.ac.jp (T. Ueda).

1046-2023/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ymeth.2005.04.006

to homogeneity in one-step chromatography compared tothe previously complicated and bothersome multi-stepchromatography. Although this in vivo method is stillwidely used by many researchers engaged in a biochemicalanalysis or practical protein application, it remains a labo-rious and long procedure. It takes several days to obtainpuriWed proteins via several experimental steps that includeDNA cloning in the vector, DNA transformation in cells,over-expression of desired protein in cells, and purifyingthe protein from cells. Because of this, in vivo productionof the protein is unsuitable for preparation of various pro-teins or production of proteins that demand trial and error,such as those that are recovered as insoluble aggregates.

In contrast, cell-free protein synthesis is rapidlydeveloping [3–5] and is simpler than in vivo methods,only requiring the addition of a template DNA ormRNA to the reaction mixture and incubation forseveral hours to yield the desired protein. Using aPCR product as a template DNA, the puriWed proteincan be obtained in just one day from cDNA. Moreover,

300 Y. Shimizu et al. / Methods 36 (2005) 299–304

reaction conditions are highly controllable: optimiza-tion of the conditions, such as the temperature oraccessory factors, can be readily achieved. These fea-tures have the potential to accelerate analysis of theprofusion of genomic information and to facilitatemedical research and drug discovery.

Cell-free protein synthesis systems are generally con-structed with cell extract prepared from E. coli, wheatgerm, or rabbit reticulocytes. This cell extract, the so-calledS30 fraction, is the supernatant of 30,000g centrifugation,and consists of all soluble cell components except macro-molecules such as cell-surface membranes and genomicDNA. As the S30 fraction contains all protein componentsnecessary to accomplish protein synthesis, simple additionof the template nucleic acids and components, such asenergy sources and amino acids, results in the synthesis ofthe desired protein. Due to the recent development of thisS30-directed, cell-free, protein-synthesis system, thismethod now generates high yields of protein; 1ml of reac-tion mixture has been reported to synthesize milligram ofprotein using dialysis membrane for the continuous supplyof substrates [5,6]. However, in this cell extract, only aminority of the factors participate in the translation reac-tion, and some of the non-participating factors inhibit thereaction (e.g., nucleases and proteases that degrade sub-strates and products). Furthermore, the complexity andincomplete knowledge of the components within the sys-tem reduces the controllability.

To address these issues, we constructed a modernhighly puriWed protein synthesis system [7] containingthe puriWed E. coli components found to be suYcient

for protein synthesis by Weissbach and coworkers aquarter century ago. Our system, termed protein syn-thesis using recombinant elements (PURE), containsHis-tagged versions of all protein factors. This greatlyfacilitates puriWcation of both the factors themselvesand also the in vitro-synthesized protein of interest.The latter is achieved by a unique “reverse” puriWca-tion procedure that rapidly and easily removes the pro-tein factors by binding to Ni+-chelating resin, leavingthe puriWed protein as the sole protein component afterremoval of ribosomes by ultraWltration. In this paper,PURE translation system protocols and applicationsare detailed.

2. The PURE translation system

mRNA is translated into protein using aminoacyl-tRNA intermediates and ribosomes consisting of dozensof proteins and three ribosomal RNAs in prokaryotes. Tocomplete translation of one open reading frame (ORF)encoded in the mRNA sequence, three reaction stepsproceed on the ribosome: initiation, elongation, and termi-nation. This is followed by a ribosome recycling step tore-initiate translation. In E. coli, several translation factorstake part in each translation step: three initiation factors(IF1, IF2, and IF3), three elongation factors (EF-G, EF-Tu, and EF-Ts), three release (termination) factors (RF1,RF2, and RF3), and ribosome recycling factor (RRF). Inaddition to this main translation reaction, three other reac-tions are necessary to facilitate protein synthesis (Fig. 1):

Fig. 1. The four main reactions of cell-free protein synthesis. The four reactions are marked with boxes and drawn schematically.

Y. Shimizu et al. / Methods 36 (2005) 299–304 301

transcription to synthesize mRNA, aminoacylation oftRNAs, and energy source regeneration. Thus, besidesthe 10 translation factors described above, T7 RNApolymerase, pyrophosphatase, 20 aminoacyl-tRNAsynthetases, creatine kinase, myokinase, and nucleo-side–diphosphate kinase are also prepared and incor-porated in the PURE system. All these protein factorsare over-expressed and puriWed in His-tagged formwithout a loss in activity. Ribosomes are preparedusing sucrose density gradient centrifugation, and thePURE system is reconstituted by also including buVer,tRNA mixtures, and substrates such as the 20 aminoacids and four nucleoside triphosphates. Reactionmixtures were originally prepared in polymix buVer,which was subsequently modiWed. The present compo-sition of the PURE system is shown in Table 1. ThePURE system is now commercially available fromPost Genome Institute Co., Ltd (Japan) as PURESYS-TEM kits.

3. Standard protocol for protein synthesis and puriWcation using the PURESYSTEM kit

3.1. Protein synthesis

First, the template for the protein of interest needsto be prepared. Either plasmid DNA, a PCR fragment,or mRNA can be used. The DNA template requires aT7 promoter sequence and ribosome binding site(Shine–Dalgano sequence) upstream of the ORF. TheORF must initiate with an initiation codon (ATG) andterminate with a termination codon (TAG, TGA orTAA). When plasmid DNA is used, a T7 terminatorsequence is also necessary downstream of thetermination codon. When mRNA is the template, thesequence should be similar to the DNA sequence butwithout transcriptional promoter or terminatorsequences.

For translation, the template is simply added to thePURESYSTEM kit components (Table 1; stored astwo separate mixes) and the mix incubated at 37 °C for1–2 h. After the synthesis, the protein product can beanalyzed without a puriWcation step. Alternatively, thequick puriWcation method described in Section 3.2 canbe used.

Examples of protein synthesis using the PURESYS-TEM are shown in Fig. 2. Each template was preparedby PCR. Protein synthesis was carried out at 37 °C for1 h in the presence of BODIPY-Lys-tRNALys (Fluoro-TectTM GreenLys in vitro Translation Labeling System;Promega). Samples were separated by SDS–PAGE andthe gel stained by SyproOrange (Amersham Biosci-ences). Synthesized proteins are indicated by arrow-heads. This result demonstrates that the PURE system,

although reconstituted solely from E. coli translationfactors, can synthesize both prokaryotic and eukaryoticproteins.

Table 1Present composition of the PURE system

SpeciWc activities of ARS and MTF were measured using radioactiveamino acids. One unit of activity was deWned as the amount of enzymethat catalyzes the formation of 1 pmol of aminoacyl-tRNA in 1 min.

Translation factors2.7 �M IF10.40 �M IF21.5 �M IF30.26 �M EF-G0.92 �M EF-Tu0.66 �M EF-Ts0.25 �M RF10.24 �M RF20.17 �M RF30.50 �M RRF

Aminoacyl-tRNA synthetases1900 U/ml AlaRS2500 U/ml ArgRS20 mg/ml AsnRS2500 U/ml AspRS630 U/ml CysRS1300 U/ml GlnRS1900 U/ml GluRS5000 U/ml GlyRS630 U/ml HisRS2500 U/ml IleRS3800 U/ml LeuRS3800 U/ml LysRS6300 U/ml MetRS1300 U/ml PheRS1300 U/ml ProRS1900 U/ml SerRS1300 U/ml ThrRS630 U/ml TrpRS630 U/ml TyrRS3100 U/ml ValRS

Other enzymes4500 U/ml MTF1.2 �M ribosomes4.0 �g/ml creatine kinase3.0 �g/ml myokinase1.1 �g/ml nucleoside-diphosphate kinase2.0 units/ml pyrophosphatase10 �g/ml T7 RNA polymerase

Energy sources2 mM ATP, GTP1 mM CTP, UTP20 mM creatine phosphate

BuVers50 mM Hepes–KOH, pH 7.6100 mM potassium glutamate13 mM magnesium acetate2 mM spermidine1 mM DTT

Other components0.3 mM 20 amino acids10 mg/ml 10-formyl-5,6,7,8-tetrahydrofolic acid56 A260/ml tRNAmix (Roche)

302 Y. Shimizu et al. / Methods 36 (2005) 299–304

3.2. Quick puriWcation method using Ni+-chelating resin and ultraWltration

This protein-synthesizing system contains a uniquecombination of two features that enables a novelmethod for the puriWcation of the synthesized protein:only those essential elements for the protein synthesisare present, and all protein factors except ribosomal pro-teins are in His-tagged form. After protein synthesis,Ni+-chelating resin (e.g. Ni-NTA agarose from Qiagen)is added to the reaction and the mix is incubated for 1 hat 4 °C with shaking. Subsequently, the resin, the resin-bound His-tagged proteins, and the ribosomes (MW»2 MDa) are simultaneously removed by ultraWltrationthrough a membrane with a 100 kDa MW cut oV (e.g.,Microcon YM100 from Millipore).

To illustrate this quick puriWcation method, DHFRprotein was synthesized, puriWed as above, run on anSDS–PAGE and stained for proteins (not RNAs). Theonly protein detected corresponded to DHFR [7].

4. Protein synthesis under two new reaction conditions that foster protein folding

4.1. Protein synthesis using a PURE system containing molecular chaperones

For the correct folding of several proteins, molecu-lar chaperones are required during and/or after transla-

Fig. 2. Synthesis of prokaryotic and eukaryotic proteins by thePURE system. The prokaryotic proteins �-galactosidase (�-Gal),dihydrofolate reductase (DHFR), DnaJ, enolase, and CAT, and theeukaryotic proteins MAP kinase, IL-8, and Ras were synthesized inthe presence of BODIPY-Lys-tRNALys. Samples were applied toSDS–PAGE and the gel was stained by SyproOrange. Proteins werevisualized by Typhoon (Amersham Biosciences). Green bands aresynthesized proteins (also marked by arrowheads) and red bands areproteins in the PURE reaction mixture.

tion [8]. These proteins form aggregates whensynthesized in the original chaperone-free PURE sys-tem [9]. However, we found that incorporating molecu-lar chaperones such as DnaK and chaperonin GroEL/GroES into the PURE system yields correctly foldedproteins exhibiting high activity. In our previousreports, single-chain Fv (scFv) protein with high anti-gen-binding activity could not be synthesized in theabsence of chaperones. In contrast, in the presence of1 �M DnaK, 0.4 �M DnaJ, and 0.4 �M GrpE (Stress-gen), scFv protein was synthesized with higher solubil-ity and binding activity [9]. Alternatively, someproteins need chaperonin for correct folding.

An example is shown in Fig. 3A, where E. coli DapAprotein became soluble when synthesized in the presenceof 1 �M GroEL and 2 �M GroES. Thus, appropriatechaperones can aid folding in the PURE system.

Fig. 3. Improved protein folding in the PURE system by addition ofchaperones or performance under oxidizing conditions. (A) Proteinsynthesis in the presence of molecular chaperones. E. coli DapA pro-tein was synthesized at 37 °C for 2 h using the PURE system in theabsence or presence of GroEL and GroES at the indicated concen-trations. After synthesis, reaction mixtures were centrifuged at20,000g for 30 min. Total (T), precipitate (P) and supernatant (S)fractions of each sample were analyzed by SDS–PAGE. Proteinswere visualized by Typhoon. Synthesized DapA protein is indicatedby the arrow. (B) Protein synthesis under oxidizing conditions.E. coli alkaline phosphatase (AP) was synthesized at 37 °C for 1 husing the original PURE system (reduced conditions) or modiWedPURE system (oxidizing conditions). After the synthesis, the reac-tion mixtures were analyzed by SDS–PAGE and the amount of APsynthesized was quantiWed by Typhoon. Enzymatic activity of APwas measured using p-nitrophenyl phosphate as substrate [21]. Theactivity and amount of AP synthesized under oxidizing conditionswere set to 100 arbitrarily units (a.u.).

Y. Shimizu et al. / Methods 36 (2005) 299–304 303

4.2. Protein synthesis under oxidized conditions

Since most protein synthesis occurs under reducingconditions, the original PURE system was also devel-oped under reducing conditions. However, proteins suchas secretory proteins that function outside the cell tendto require the formation of correct disulWde bonds toachieve their activities. Accordingly, protein synthesisunder oxidative conditions is also required as well as theone under reduced condition. Because the PURE systemis a reconstituted translation system, conditions can eas-ily be adjusted from reducing to oxidizing by addition ofoxidized glutathione instead of dithiothreitol. Further-more, addition of enzymes such as protein disulWdeisomerase aids formation of the correct disulWde bonds.A PURE system kit suitable for synthesizing proteinsthat contain disulWde bonds in their tertiary structure iscommercially available as PURESYSTEM S–S kit (PostGenome Institute Co., Ltd, Japan).

A protein containing two disulWde bonds, alkalinephosphatase (AP), was synthesized using PURESYS-TEM S–S kit and the original PURE system, and thespeciWc activities of the synthesized AP proteins werecompared. Though the AP from the original PURE sys-tem had low activity, AP from the PURESYSTEM S–Skit had several folds higher activity (Fig. 3B). It shouldbe noted that purchased AP is several folds more activethan that from the S–S kit, so we are engaged in furtheroptimization of the kit.

5. Applications

Besides synthesizing active proteins, the puriWed pro-tein-synthesis system has many applications which uti-lize its high controllability. Cell-free systems providegreater Xexibility for incorporation of an unnaturalamino acid at a speciWc residue of a synthesized proteinusing a suppressor-tRNA charged chemically or enzy-matically [10,11]. This employs nucleic acid templateswith a termination codon inserted inside the ORF of thegene to be read through by a suppressor-tRNA.

Protein selection from large libraries by methods suchas ribosome display [12], in vitro virus [13], or mRNAdisplay [14] is also facilitated by using a cell-free proteinsynthesis system. All these selection techniques requirethe linking of genotype (mRNA) to the phenotype (syn-thesized protein) in the translation mixture for the subse-quent ampliWcation of the genetic information of theselected protein. For this linkage, ribosome display uti-lizes the formation of a peptidyl-tRNA/ribosome/mRNA complex whereas in vitro virus and mRNA dis-play utilize the covalent formation of peptidyl-puromy-cin–mRNA by using an mRNA–DNA–puromycinfusion as the template. Because these selection methodsoperate entirely in vitro, they oVer advantages over simi-

lar in vivo selection methods [15,16] such increasedlibrary sizes and ease of mutagenesis.

The PURE system, which is more easily controlledthan the conventional S30 system, is suitable not onlyfor the analysis of the molecular mechanism of prokary-otic translation [17,18] but also for extending the appli-cations described above. In our previous report, highlyeYcient amber suppression was enabled by omission of aspeciWc release factor to avoid competition between therelease factor and suppressor-tRNA [7]. Omission ofthe release factor may also aid stalling of the ribosome atthe termination codon to form a peptidyl-tRNA/ribo-some/mRNA complex necessary for ribosome display.Furthermore, pure translation systems lack mRNA-degrading nucleases, facilitating our novel screening sys-tem for mRNA variants that bind to their PURE systemtranslation products [19] and also “pure translation dis-play” [20].

6. Concluding remarks

Here, we described the unique features, methodologyand applications of various PURE systems, two ofwhich have now become widely available as kits.Though their protein synthesis capabilities are broad,they are obviously less sophisticated than living cellswhich have various sub-systems besides our core proteinsynthesis components to facilitate synthesis of activeproteins. For this reason, some test proteins were syn-thesized in an insoluble form in the PURE system. Inthis paper, we demonstrated that addition of molecularchaperones enables synthesis of certain proteins withoutformation of insoluble aggregates. In addition, proteinsynthesis under oxidizing conditions resulted in the for-mation of correct disulWde bonds and catalytic activity.These data illustrate the advantage of translation reac-tions that can be tailored to individual target proteinsusing several sub-systems. In our previous report [7], wementioned diYculties with mRNA-directed translation,presumably because of the absence of RNA helicases.We tested helicases, such as the Dead-box protein, andalso SrmB protein that is reported to stabilize mRNA,with little eVect on the translation eYciency, suggestingother sub-systems may be necessary to accommodatemRNA templates. Furthermore, we tested whether ornot the continuous dialysis system would give higherprotein yields. It was unable to prolong the translationreaction, again suggesting that other sub-systems maybe helpful.

Beyond synthesizing the active proteins asdescribed in this paper, the PURE system is set toenable the new applications described in Section 5.Further development of the PURE system shouldopen up additional Welds of protein research in thenear future.

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