Towards the construction of Escherichia coli cell-free protein

synthesis system platform

Sara Alexandra Peca de Sousa Rosa

Instituto Superior Tecnico, Lisboa, Portugal

July 2015

Abstract

Cell-free protein systems are described as the in vitro expression of recombinant proteins withoutthe use of living cells. This approach uses a cell lysate containing a wide array of biological andchemical components for transcription, translation, protein folding, and energy metabolism; all requiredto directly synthesise the target protein. However, there are some problems when using these systems:the ability to reliably synthesize any biologically active protein in a universal platform, the lack of acost-effective and scalable platform, and the inability to carry out humanized glycosylation patterns.The main aim of this work is to construct a robust and cost effective platform for in vitro proteinsynthesis in a coupled transcriptiontranslation system.

In this work, a DNA template was design and purified. The DNA template was purified using analkaline lysis and hydrophobic interaction chromatography. This method allows us to produce in higherquantity and lower cost. The tRNA purification was achieved by extracting the nucleic acids by phenolextraction, separate the DNA with sodium acetate, and remove the remaining contaminants by anionexchange chromatography. When producing the S30 lysate, it was not possible to achieve the proteinconcentration necessary to have a highly active lysate.

Although the main goal of creating a reproducible E. coli cell-free protein synthesis system platformwas not achieved, some major steps were taken towards this goal. In the future, one of the priorities isto improve the lysate production method so that we get a better yield from the cell-free system.Keywords: Cell-free systems, Escherichia coli, S30 lysate, tRNA purification, Plasmid purification

1. Introduction

Cell-free biology is defined as the activation of com-plex biological processes without using intact livingcells [5, 19]. The metabolism can focus cellular re-sources towards an exclusive and defined objective.The removal of physical barriers enables the directaccess to the inner working of the cell: allowing easysubstrate addition, product removal, system moni-toring, and rapid sampling. Furthermore, with theremoval of genomic DNA, there is no longer theneed for genetic regulation (and cell viability is alsoremoved). In short, cell-free biology decrease thedependence on cells and increases the engineeringflexibility [3].

The most prominent and developed exampleof cell-free biology is cell-free protein synthesis(CFPS) [4]. As the name implies, CFPS is de-scribed as the in vitro expression of recombinantproteins, without the use of living cells. It uses a celllysate containing a diverse array of biological andchemical components for transcription, translation,protein folding and energy metabolism required todirectly synthesise the target protein. The lysatescan –theoretically– be created from any cell source

or origin and can be reconstituted for biosynthesisof proteins by adding exogenous genetic informa-tion. The added substrates include amino acids,nucleotides, DNA or mRNA template encoding thetarget protein, energy substrates, cofactors, andsalts. As in a fermentation reaction, the cell-freeprotein synthesis production continues until one ofthe substrates (e.g. ATP or cysteine) is depleted asthe by-product accumulation reaches an inhibitoryconcentration.

Cell-free protein synthesis systems are becomingincreasingly more popular due to them advantagesin the protein production process when comparedto the in vivo approach. They are considerablyfaster, because the process does not involve trans-fection or cell cultures, nor does it require a difficultdownstream purification. Moreover, only the de-sired protein is produced so there are no expensesin the production of proteins and metabolites in-volved in the cell maintenance and growth. It alsoreduces the effect of toxicity, because there is noneed to maintain the cell viability (it only main-tains the metabolisms required to the production).

With cell-free systems, one can influence the com-

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plex reaction network directly, by adjusting differ-ent supplies for the optimal production. Catalystsand reagents can be added or removed: such as com-plex enzymatic subtracts, cofactor or non-naturalreagents, and energy supplier. Template or PCRproduct concentration can be adjusted to optimalproduction. Another advantageous feature is thepossibility to monitor the complex reaction networkdirectly, because it easy to acquire samples, andenables on-line monitoring. Duo to the absence ofcell membranes, there are no barriers to access theproduct. There is no need for transporters, and itsimplifies the product harvest and the monitoringof the product levels.

In recent years, there has be growing interest tomatch Escherichia coli based cell-free protein syn-thesis systems to the increasing demands for sim-ple, inexpensive, and efficient protein production.However, the current protocols of these systems areexpensive and time consuming. For example, a sub-stantial portion (more than 30%) of the costs ofpreparing a cell-free synthesis reaction is attributedto the preparation of the lysate [14]. Therefore,the development of more economical procedures forthe preparation of cell-free components is crucial forexpanding the application of cell-free protein syn-thesis. The main objective of this work is to con-struct a robust and cost effective E. coli cell-freesystem platform. The work focused the prepara-tion of three main components of this system: Thetemplate, total tRNA and lysate.

A highly purified DNA template have a ma-jor influence in the protein yield. This is usuallyachieved using available commercial kits. In thiswork, we test alkalyne lysis and Hydrophobic Inter-action Chromatography (HIC) for plasmid purifi-cation. Total tRNA purification is also attempted.The addition of exogenous tRNA to the cell-free sys-tem not only increases the protein yield, but it al-lows to control the system through changing tRNAcomposition [8] and facilitates in incorporation ofunnatural amino acids. Here, two protocols basedon phenol extraction, precipiation and Anion Ex-change Chromatography (AEC) are tested.

Preparation of S30 lysate is one of the most im-portant steps for a successful construction of a cell-free system. In this step is important to chosethe adequate E. coli strain, and the mechanism ofcell disruption. Here, BL21(DE3) strain and beadmilling as lysis mechanism were tested for the lysateproduction. Besides testing the produced lysateswith the cell-free production. Finally, the construc-tion of a cell-free system using Cytomimic as theenergy system, and other cell-free system variableswere studied: magnesium and plasmid concentra-tion, and lysate volume.

2. Materials and Methods2.1. Template preparationThe pEXP5-NT/GFP was produced in this study.The GFP gene was isolated from pVAXIGFP-BHGIand inserted in pEXP5-NT vector. The pEXP5-NTwas obtain by excision of CALML3 gene. Besides,it contains a N-terminal tag (His-tag), a bacterio-phage T7 promoter, a ampicilin resistance gene forselection in E. coli, and a pUC origin.

2.2. Plasmid production and purificationA cell bank containing BL21(DE3) with pEXP5-NT/GFP was inoculated in a 500mL kitasato flaskwith 100 mL of TB medium (pH 7.4) (12g×L−1 oftryptone (BD), 24g × L−1 of yeast extract (Hime-dia), 4mL × L−1 glycerol (JMGS)) supplementedwith ampilicin (Sigma) (100µg × mL−1). The in-oculum was grown until it reached an OD600 of 3nm in a orbital shaker at 37oC and 250 rpm. A5 litre Biostat B fermenter, containing 4L of TBmedium (12g×L−1 of tryptone, 24g×L−1 of yeastextract, 4mL×L−1, 17mM KH2PO4 and 0.72mMK2HPO4 (Panreac)) at pH of 7.4, supplementedwith the respective antibiotic, was inoculated withthe previous culture. The culture was fermentedovernight with a oxygenation rate of 30%.

The cells were disrupted by alkaline lysis. Thepellets were resuspended with P1 buffer (50 mMglucose (Merck), 25mM tris-HCl (Eurobio), pH 8.0,10 mM EDTA (Sigma), pH 8.0). The same amountof P2 buffer (0.2N NaOH (Merck), 1% (m/v) SDS(Merck) was added to the mixture and then fol-lowed by gentle homogenization. The resultant so-lution was left to rest at room temperature for 10minutes. To stop the reaction, P3 (3M potassiumacetate (Panreac), acid acetic (Fisher)) was addedto the mixture. After gentle homogenisation, themixture was placed on ice for 10 minutes. The neu-tralised alkaline lysate was centrifuged (Sorvall RC-6 Plus, SS34 rotor) twice at 20000 × g and 4oC for30 minutes. Between centrifugations the pellet wasdischarged.

To supernatant obtained was added 0.7 volumeof isopropyl alcohol (Sigma). After homogenisation,the mixture was left for 2 hour at −20oC. Then,it was centrifuged at 2000 × g and 4oC for 30 min-utes. The supernatant was discharged and the pel-let was resuspended in 10 mM Tris-HCL (pH 8).Ammonium sulphate was added to the solution in aconcentration of 0.33g×mL−1 and after homogeni-sation, the resulting solution was left on ice for 15minutes. Then, it was centrifuged at 1300 rpm and4oC for 30 min. The supernatant was collected.

Plasmid was purified by hydrophobic interactionchromatography (HIC) in and AKTA 100 Purifiersystem, using a Phenyl Sepharose 6 fast flow (highsub) column (Tricorn 10/100 column, GE Health-care). First, the column was equilibrated with 3

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column volumes of 1.5M ammonium sulphate (Pan-reac) in 10 mM Tris-HCL (pH 8). 1mL of the sam-ple was injected into the column. To remove theunbounded material, the column was washed with2 column volumes of 1.5M ammonium sulphate in10 mM Tris-HCL (pH 8). The run was performed ata flow of 1 mL× minute−1. The elution was madewith 3 column volumes of 10 mM Tris-HCL (pH 8).

In order to exchange the buffer where the puri-fied plasmid was accommodated, a size exclusionchromatography (SEC) was performed. The samecolumn of HIC purification was used. 3 column vol-umes of Tris-HCL (pH 8) were use to equilibrate thecolumn. 1 mL of purified plamids in 1.5M ammo-nium sulphate in 10 mM Tris-HCL (pH 8) was in-jected and the column was washed and eluted with3 colunm volumes of 10 mM Tris-HCL (pH 8) ata flow of 1 mL× minute−1. 1.5 mL fractions werecollected.

2.3. tRNA purificationThe extraction of the cellular RNA was done byphenol extraction. All the buffers use were preparedwith autoclaved water. All centrifugation were per-formed at 4oC, unless specified. The cells were re-suspended in 25 mL of 1.0 mM TrisHCl, 10 mMMgCl2, pH 7.2. The same amount of phenol sat-urated in the same solution was added and the itwas vigorous mixed for 2 hours. Then, it was cen-trifuged at 15000 × g for 30 minutes.

After centrifugation, two different phases wereobserved: a fluid and transparent phase at top andone turbid was viscous at he bottom. The fluidone was collect and 0.1 volumes of 20% potassiumacetate and 2 volumes of 70% was added. After ho-mogenisation, the mixture precipitated overnight at-4oC. The pellet was collected by centrifugation.

In order to remove the contaminants, such asDNA and rRNA, separation with a low pH bufferand purification by anion-exchange chromatogra-phy [9] was tested. 1 volume of 0.3 M sodium ac-etate, pH 5 and 0.5 volumes of isopropyl alcoholwere used to resuspend the pellet. The mixture wasvigorously stirred at room temperature for 1 hourand the precipitate was removed by centrifugationat 15000 × g and 20oC for 30 minutes. The super-natant was cooled to 4oC and 0.5 volumes of iso-propyl alcohol was added. After 2 hours at −20oC,the precipitate was collected by centrifugation at15000 × g for 30 minutes. The precipitate was dis-solved in 10 mM of Tris-HCl (pH 8).

The tRNA was further purified by Anion-exchange chromatography in AKTA 100 Purifiersystem, using a DEAE Sepharose 6 fast flow (GEHealthcare) column (Tricorn 10/50 column, GEHealthcare). First, the column was equilibratedwith 3 column volumes of 10 mM Tris-HCl (pH8). 1 mL of the tRNA samples was injected to the

column. To remove the unbounded material, thecolumn was washed with equilibration buffer. Theelution was made with a step of 30% 1 M NaCl, 10mM Tris-HCl (pH 8) and 70% with 10 mM Tris-HCl(pH 8).

2.4. S30 Lysate preparationFor the production of S30 lysate, BL21(DE3) strainwere used. Bead mill was chosen to perform a me-chanical disruption was bead mill [18]. In figure 1 adiagram of the lysate processing used is represented.

2×Y T medium (16g×L−1 Tryptone, 10g×L−1

yeast extract,5g×L−1NaCl ) (pH 7.4) and was usedin the pre-inuculum. 2× Y TPG ( 16g×L−1 Tryp-tone, 10g × L−1 yeast extract,5g × L−1NaCl ,22mM sodium phosphate monobasic (Sigma), 40 mMsodium phosphate dibasic (Panreac), 100 mM glu-cose) was used as inoculum. The cells were har-vested in mid-log phase. Cell were then washedwith 100 mL of 1×S30 buffer (10 mM Tris acetate,14 mM magnesium acetate, 60 mM potassium ac-etate) and centrifuged again in the same conditions.The pellet was were weighed and stored at −20oC.

The pellet was resuspended with 1 mL of roomtemperature S30 buffer per gram of cells. Two con-centrations of cell beads were mixed in the solution:3 and 5 grams of autoclaved 1 mm beads × grams ofcell. The cells were lysed in a bead mill (Vibrogen-Zellmhle, Edmund Bhler). For 1000 mL of cell cul-ture, 4 cycles of 1 minutes (30 seconds on, 30 sec-onds on ice) were used and, for the 2000 mL culture,6 cycles of 1 minute (30 seconds on, 30 on ice). Toseparate the beads and cell debris from the lysate,a centrifugation was performed at 30000 × g for 30minutes. The collected supernatant was incubatedin a orbital shaker at 37oC for 80 minutes. Thenthey were centrifuged at 18000 × g for 20 minutes.The final supernatant was aliquoted and stored at−80oC.

2.5. Cell free protein synthesisA Expressway Cell-Free E.coli Expression systemkit (Invitrogen) was acquired for test the plasmidand lysates produced. This kit contained all thecomponents needed to produce and a the pEXP5-NT/CALML3 for a positive control. Besides thekit, a system based on Cytomim system [22] wastested, and optimisation attempts were made.

The reaction was performed in a sterile 1.5 mLeppendorf tube at 37oC in a orbital shaker at 300rpm for 6 hours. The final volume of each reac-tion was 25µL, and the reagents were added in theamounts represented in table 1.

After the first 30 minutes of reaction, a Feedbuffer was added to each reaction, containing thereagents represented in table 2.

A reaction based on th Cytomim system wasperformed in a sterile 1.5 mL eppendorf tube con-

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Cell Culture(Shake Flask)

Cell disruption(Bead mill)

Centrifugation(1X 30,000 g, 30 min, 4ªC)

Run-off reaction(37ºC, 80 min, 250 rpm)

Centrifugation(1X 18,000 g, 30 min, 4ºC )

Figure 1: Steps of the s30 lysate processing.

Reagents Volume (µL)E. coli sly D- Extract 52.5× IVIPS Reaction Buffer 550 mM Amino Acids (-Met) 0.375 mM Methionine 0.25T7 Enzyme Mix 0.25DNA template 0.5Dnase/Rnase-free Distilled Water 1.2Total volume 12.5

Table 1: Reagents used to perform cell-free proteinsynthesis from Expressway kit and the respectivevolumes in µL

Reagents Volume (µL)2× IVIPS Feed Buffer 6.2550 mM Amino Acids (-Met) 0.375 mM Methionine 0.25Dnase/Rnase-free Distilled Water 5.7Total volume 12.5

Table 2: Feed buffer reagents from Expressway kitand the respective volumes in µL

taining 25µL of reaction. Beforehand, two bufferswere prepared: 10× Salt solution, containing 1.3M potassium acetate, 100 mM ammonium acetate(Sigma), 80 mM magnesium glutamate, and 10×Master mix, containing 12 mM ATP, 8.5 mM GTP,8.5 mM CTP, 8.5 mM UTP, 340µg ×mL−1 folinicacid, 1.7mg×mL−1 E. coli tRNA. Besides, the fol-lowing reagents were prepared: 1 M Sodium pyru-vate (Fisher), 100 mM Sodium Oxalate (Fisher),100 mM Spermidine (Fisher).

The reaction was performed at 37oC in a orbitalshaker at 300 rpm for 5 hours. The reagents concen-trations and volumes used are represented in table3. The plasmid and magnesium concentrations andthe lysate volume was further optimised.

3. Results3.1. Plasmid purificationHydrophobic interaction (HIC) is a method ofchoice when pDNA purified. It separates the pDNAbased on the difference in surface hydrophobicitybetween the supercoiled plasmid and the impurities(RNA, endotoxins and proteins). It is a purificationprocess that achieves high purity and considerable

amounts of plasmid DNA.First, the samples is loaded and the column is

washed with high salt concentration. In this step ainteraction between the hydrophobic functional re-gions of the solutes and hydrophobic function takesplace [20]. Since the plasmid is double stranded, itcontains a week hydrophobic nature, and it is elutedwhile the impurities are retained in the column. Be-cause HIC binding process in more selective thanthe elution process, the plasmid separation dependson the optimization of the start buffer conditions,such as the salt used, its concentration and purity.

In this case, 1.5 M ammonium sulphate was cho-sen as a binding buffer due to: high salting-out abil-ity, high solubility varying little in a range of 030oC,stability up to pH 8.0, and low cost [1]. Besides itwas already successfully implemented in the labo-ratory. For the elution buffer, 10 mM Tris-HCl pH8 was chosen. 1 mL of the sample was injected to acolumn equilibrated the binding buffer. The columnwas washed with 2 column volumes of the bindingbuffer and eluted with 3 of 10 mM Tris-HCl ph8.

The figure 2a presents the chromatogram corre-spondent to the pEXP5-NT/GFP purification bythis method after the injection (volume 0). Thefirst peak after injection may corresponds to thepurified plasmid. The second peak appears con-jugated with a peak in conductivity. This maymeans that the peak is due to the increase of saltin the flowtrough and not any contaminant. The fi-nal peak correspond to the contaminants and startswhen the conductivity decrease. When conductiv-ity decreases, the salt concentration present on theflowtrough also decreases, and the hydrophobic ar-eas of the contaminants stop to interact with thematrix and are eluted.

Figure 2b, shows a 1% agarose gel with selectedfraction of the pEXP5-NT/GFP purification chro-matography from figure 2a. The first 4 fractionscorrespondents to the first peak they present pureplasmid as expected. It is also observed that thesecond peak is indeed due to the increase of salt inthe flowtrough. The final peak contains RNA. Itis noted that the salt concentration is so high thatthe plasmid bands are not well defined and it is notpossible to identify the plasmid isoforms.

3.2. tRNA purificationAmino acids are activated for peptide bond forma-tion by amino acyl tRNA synthetases that are spe-

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Reagents Stock concentration Reaction concentration Volume (µL)10× Salt Solution 10× 1× 2.510× Master mix 10× 1× 2.550 mM Amino Acids (-Met) 50 mM 2 mM 0.675 mM Methionine 75 mM 2 mM 0.5Sodium Pyruvate 1 M 33 mM 0.83Sodium Oxalate 100 mM 4 mM 1Spermidine 100 mM 1 mM 0.25T7 RNA polymerase - - 1Lysate - 24% 2.1Plasmid Template 0.5 µg × µL−1 33 µg × µL−1 1.7Dnase/Rnase-free Distilled Water - - 8.12Total volume - - 25

Table 3: Reagents for 25µL reaction kit, stock and reaction concentrations and the respective volumesused in µL

cific for the amino acid substrate and the cognateset of tRNAs, covalently linking the residue to the3’-terminal adenosine of the tRNA [16]. For proteinsynthesis occurs, all 20 amino acids must presentin amounts super-stoichiometric with the amountof protein to be formed, in addition to catalyticamounts of all 20 amino acid tRNA synthetases andthe full complement of tRNAs.

The addition of exogenous tRNA to the cell-freesystem not only increases the protein yield, butit allows to control the system through changingtRNA composition [8] and facilitates in incorpora-tion of unnatural amino acids.

To this work, total tRNA with amino acids at-tached to the terminal adenosine groups were puri-fied. We attempted to separate RNA from pDNAand rRNA with low pH solution was used to precipi-tate pDNA and an anion-exchange chromatography(AEC) was used to remove the remaining contami-nants.

First, a phenol extraction method was performed.By adding a phenol saturated buffer to the cell mix-ture, the permeability of the cell wall will increase,and small molecules, such as tRNA, can diffuse outof the cell. This way cell lysis is avoid. Besides, itallows separate the nucleic acids from the proteins.Since phenol and the buffer are immiscible, theyform a two phases when centrifuged. Phenol is lesspolar than water, and since nucleic acids are polar,they will stay soluble in the water. This means thatwhen phenol is mixed with water, they do not dis-solve in the phenol ant they remain in the buffer.However, proteins are composed by polar and non-polar amino acids. Usually, when protein are ex-posed to a less polar solvent, the folding changesand the less polar residues, which usually are insidethe protein structure, will interact with phenol andare forced to the outside. This means that the pro-teins are denatured and they will stay in the phenol

phase. Since the top phase, containing the nucleicacids, is collected, most proteins will be removed.

After phenol extraction and ethanol precipita-tion, the pellet was resuspended with 0.3M sodiumacetate (pH 5.2) and 0.5 volumes of isopropyl al-cohol and incubated at room temperature. In thisstep, pDNA and large molecular weight RNA willbe precipitated [9]. The tRNA and remaining con-taminants were precipitated with 0.5 volumes of iso-propyl alcohol.

In the figure 3 a 1.2% agarose gel containing sam-ples of the purification is observed. The 3 1 and 2contain samples of the recovered pellet after phe-nol extraction and ethanol precipitation. 3 3 is asample of the recovered pellet after the purificationwith 0.3 M sodium acetate at ph 5.2 method. Withis method, not only the pDNA was removed butalso amost all the other RNAs.

tRNA was further purified with an anion ex-change chromatography. The resin used was DEAEsepharose and as buffers 10 mM Tris-HCl (ph 8)(equilibrium) and 1M NaCl, 10 mM Tris-HCl (ph8) (elution). An anion exchange chromatographyis a process that separate according to moleculescharge. The resin is coated with a positivelycharged counter-ions and will bind to negativelycharged molecules. In the equilibrium phase, allcations are bonded to counter ions present in theequilibration buffers. When the sample is injected,negatively charged molecules will bind to the resin.The unbounded molecules are washed out. The elu-tion is usually perform with a gradient to increaseof ion strength. The molecules are disordered ac-cording to the number of negativity charged groups.In this case, the charge phospahte from tRNA willbind to the matrix. When the ion strength exceedthe strength of the tRNA it will detach and elutedfrom the column.

In this case, the elution step was set to be with

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(a)

(b)

Figure 2: HIC chromatogram of the pEXP5/GFPpurification and a 1% agarose gel of the selectedfractions. 2b) HIC chromatogram. The X axiscorrespond to the volume of the flowtrough, andthe double y axis to the absorvance at 254 nm(green line) and the conductivity of the flowtroughin mS/cm (blue line). 2a) A 1% agarose gel of theselected fraction of pETGFP purification HIC chro-matography. 1 to 4 correspond to the first peak(pure plasmid). 7 and 8 correspond to the secondpeak. 9 to 12 correspond to the final peak (con-taminants). 13- Feed sample. M-NZYDNA LadderIII.

45% of 1 M NaCl buffer. The figure 4 shows theAEC chromatogram of tRNA purification with a45% step elution and a 1.2% agarose gel of the se-lected fractions. In the chromatogram (figure 4a) itis observed that 2 two peaks: the first correspond-ing to contaminants, and the second to the tRNA.This was verified with in the 1.2% agarose gel (fig-ure 4b).

3.3. S30 lysate preparation

The first step to prepare the S30 lysate is to chosethe E. coli strain. The first cell free systems usedstrains that lacked a major Rnase, (i.e. rna and/orrne mutations) such as MRE600 and A19. This mu-tations increase mRNA half-life [10]. Besides, thestrain should be deficient in OmpT and lon pro-tease activities to reduce proteolyic degradation ofthe target protein.

In this study, the BL21(DE3) strain was cho-sen to produce the S30 lysate. First, it presents aλprophage carrying the t7 RNA polymerase gene

Figure 3: A 1.2% agarose gel containing differ-ent steps of tRNA purification. 1 and 2-Samplesthe supernatant of the precipitation with ethanol.3-Pellet sample after the purification with 0.3 Msodium acetate. M-NZYDNA Ladder III.

and a lacIq. This allows the protein produc-tion from plasmid containing T7 promoter, such aspEXP5-NT/GFP, pEXP5-NT/CALM3 and pET-GFP. It also lacks OmpT endoproteinase, lon pro-tease and a major RNase, making them one of thebest strains for the extract lysate. Besides, thisstrains don’t have a tight control for the produc-tion of T7 RNA polymerase, which means that evenwithout induction, the strain has a basal expression.This way, some amount if T7 RNAP will be presentin the lysate.

However, in recent studies a BL21-derivativestrain containing extra copies of argU, ileY, andleuW tRNA genes, which encode tRNA speciesthat recognize the arginine minor codons AGA andAGG, the isoleucine minor codon AUA, and theleucine minor codon CUA, respectively (i.e. BL21codon-plus strains, Stratagene, or Rosetta strains,Novagen) has been used. Results showed thatlysates has nore 50% activity for cell-free proteinsynthesis [10].

Other important aspect that influences proteinsynthesis in a cell free system is the cell growth. Thegrowth rate of the culture determines the riboso-mal content of the extract [21] ,so a medium whichsupports rapid growth ( µ>0.5h−1) should be usedfor best results. Usually, 2× YT supplement withglucose and inorganic phosphate is the medium ofchoice. because it produce better lysated and longercell-free reactions. Studies demonstrated that, al-though the cell growth rate is similar between 2×YT and 2× YTPG, the lysates prepared from withcells gronw in the last has lower phosphatase activ-ity [13]. Besides, the glucoses added is used from amore efficiently regeneration of ATP, due to enrich-ment of glucose-metabolizing enzymes. It was also

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(a)

(b)

Figure 4: AEC chromatogram of tRNA purificationand 1.2% agarose gel of the selected fractions. 4a)AEC chromatography with a elution step of 45%NaCl buffer. The X axis correspond to the vol-ume of the flowtrough, and the double y axis to theabsorvance at 254 nm (green line) and the conduc-tivity of the flowtrough in mS/cm (blue line) andthe percentage of 1M NaCl buffer in the flowtrough(%B) (orange line). 4b) A 1.2% agarose gel contain-ing: 1 to 5 Fractions from the first peak; 6 to 9-Theselected fractions corresponding to tRNA peak; 11-Feed sample. M-NZIDIA Ladder III.

demonstrated that the regeneration of ATP due toglucose leads to a higher rate of protein synthesisin a cell free system.

The second and most important step in the S30lysate preparation is the biomass processing. Thefirst step is to add S30 buffer to the biomass. Mostprotocols call for the addition of 1 mL per gram cellpaste. This value can vary, since use more bufferlead to a more dilute cell lysate an improve clarifi-cation. The choice of volume used must be the mostconvenient for the lysis method used. However, theoptimum volume of extract used for cell-free reac-tions will depend on the cell dilution factor. If morebuffer is used to resuspend the cells, more extractwill be needed in the reaction. In this case, a 1 mLper gram of cell paste was chosen to use.

After resuspension, the cell must be lysated. Thestep is a key operation on lysate preparation. Thefirst methods usually used a fresh press to pro-duce the lysate. Nowadays the most common pro-tocols use a specializes high-pressure homogenizer.

However, this equipments are expensive, so recentlythere is effort to develop methods to lysate the cellsusing a less expensive and more common equip-ment [14]. There are already studies that use beadmilling, bead vortexing [17], and a sontication studyfrom this year.

In this study, a bead mill was the equipment cho-sen for this step. The method used was based onpublished studies [18]. The first batch produced, 3grams of beads per grams of cell were used and, inthe next 5 batchs, 5 grams. The first 3 batches wereproduced with cell paste corresponding of 1L of cellculture. In the last three, 2L of cell culture wasused. For the 1L of cell, the cycles of bead millingwere optimised and the final protocol obtained was4 cycles of 30 seconds on, 30 seconds on ice. For the2L of cell culture the protocol used was 6 cycles of30 seconds on, 30 seconds on ice.

After cell disruption, the lysate was centrifuged.Usually, 2 sequential centrifugations are preform at30000× g, for 30 minutes. Recent studies indicatethat centrifugation can be done at a lower g-forceand for less time [12]. In this study, the centrifuga-tion was performed just one time at 3000× g for 30minutes.

The next step is a reaction named run-off. It wasthought that this step allowed the ribosomes to fin-ish translating their current mRNA substrate to bereleased for in vitro translation of the mRNA of in-terest [6]. The usual reaction os performed with theaddition of several expensive components. In a re-cent study, this reagents were omitted, simplifyingthe process and the cost [17]. An added benefit ofthe empty runoff is that the absence of the reagentsprevents unnecessary dilution. However, withoutthis addition, the 70S ribosomes do not dissociateinto subunits. In this study, he runoff reaction wasperform without the addition of reagents, at 37oCfor 80 minutes. Most lysate preparation proceduresinclude extensive dialysis after the runoff reaction.Typically this is done at 4oC for four 45 minutesperiods versus 20 volumes of S30 buffer (changedevery 45 minutes) using 68000 Da molecular weightcut-off dialysis tubing. Recent studies showed littleif any benefit of the dialysis step for cell-free reac-tions [17]. In this study, the first 3 lysates weredialysed against 20 volumes of S30 buffer overnightusing a 12-14000 Da dialysis tubing.

After preparation, the S30 lysate must be storedin aliquots at −80oC sin ce higher temperatures canreduce the extract activity. Also, the lysate vol-ume is usually obtained in small quantities. In mostcases 2 mL of lysate is obtained from 1 L cell cul-ture. Other important aspect is the protein concen-tration. Usually, the lysate must contain between27-30 mg×mL−1 of protein [17]. However, extractcharacteristics vary from batch to batch.

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The table 4 represent the protein concentrationfound in the 6 lysates as well as a summary of theprocedure used in each of them. The final entry cor-responds to the protein concentration on the lysateof Invitrogen cell-free kit. Not much conclusionscan be taken from this. None of the lysates havethe desired protein concentration. However it seemsthat the last one is more closed that the other. Thisone was produced with cells not containing plasmid.This low protein concentration may be due to thelysis equipment used. The bead mill available in thelab did not allow to chose the velocity as the onesdescribed on other studies. Besides the cell werelysed at once, and then transferred to the centrifu-gation tubes. Since the solution was very viscous,and during the transference some of solution waslost. Other studies did the lysis and centrifugationin the same tubes [18]. To understand better if thelow protein concentration is due to the lysis equip-ment, another lysis method should be tested, suchas high-pressure homogenizer. Since it widely usedfor this process it would be easer to compare theresults.

4. Cell-free protein synthesisThe first step towards the construction of a cell-free platform was to test pEXP5-NT/GFP. In the-ory, this plasmid has all the components neces-sary to be used as a expression vector in transcrip-tion/translation E.coli cell-free system. The GFPproduction was confirmed by measuring the fluores-cence of active GFP using a VersaFluor fluorome-ter with EX490/10 excitation filter and EX510/10emission filter (Bio-rad). The GFP concentrationwas calculated by a concentration curve measuredwith pure GFP protein produced from BL21(DE3)cells containing pEXP5-NT/GFP. Using this kit, wewere able to produce 9.25 +- 5.79 µg × µL−1. Thehigh standard deviation may be due to operationerrors since we are dealing with small volumes.

The 6 lysates produced were also tested with Ex-pressway cell-free kit. As positive control, the lysatefrom the kit was used. The tests were perform atthe temperature recommended for the kit (37oC).The figure 5 contains the obtained results. It seemsthat nether of the lysates produced protein with acomparable amount of the kit lysate. This was nota suprise for a number of reason: first, the kit isoptimised for the production with the lysate con-tained in the kit; second: The protein concentra-tion present in the lysates was sensitively lower thanthe present on kit lysate. The last lysate showeda slightly hig had a higher protein concentration.Taking into account this results and the lysate vol-ume achieved, the lysate 6 could be chosen to beused in the next studies.

After choosing the template and the lysate, theenergy system was explored. The Cytomim system

Figure 5: A histogram containing the average rela-tive flurescence (RFU) of the produced GFP in the6 lysates and the lysate from the kit.

was chosen to used in this cell-free system. Thisenergy system expores the activation of oxidativephosphorylation. In the presence of thiamine py-rophosphate (TTP) and flavin adenine dinucleotide(FAD), acetyl phosphate is generated through thecondensation of pyruvate and inorganic phosphate[15]. Then, the acetyl phosphate is catalysed byendogenous acetyl kinase in the E. coli S30 lysate.This system requires the presence of oxygen for thegeneration of acetyl phosphate and produces H2O2

as a byproduct, but it is sufficiently degraded byendogenous catalyse activity [11]. The Cytomimsystem requires inverted membrane vesicles to per-form oxidative phosphorylation. These vesicles areformed during the lysis step, usually a high pressurehomogenization, and remain in the final lysate. TheGFP obtained was measured by fluorescence of ac-tive GFP. 54.50 +- 3,25 relative fluorescence unitsof GFP were obtained.

This system was further optimised. The mostsensitive variable of a E.coli is the magnesiumconcentration [22] [18]. The standard conditions(8mM) should provide a acceptable activity, nev-ertheless the different concentrations concentrationshould be tested. In this case, the magnesium con-centration was varied from 2 to 12 mM in incre-ments of 2mM. Other variable optimised was theplasmid concentration. Finally, tanking into ac-count that the lysate used has a low protein con-centration, increasing the volume used was alsoscreened.

The figure 6 shows the graphics correspondent tothe screens. The 6a represents the active GFP pro-duced, in relative flurescence units, with the incre-ment of magnesium concentration. It seems thatthe optimal Mg2+ is between 6 and 8 mM. How-ever, this results can only be applied to this lysatebatch. For every lysate, magnesium concentrationmust be optimised [22]. In case of plasmid concen-tration optimisation (figure 6b), it seems that from33 µg × µL−1, increasing the concentration doesnot has much effect in protein production. This

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Lysate Cell culture Bead mill lysis Dialysis Protein concentration (mg ×mL−1)1 1L 3 grams and 4 cycles yes 9.782 1L 5 grams and 4 cycles yes 14.663 1L 5 grams and 4 cycles yes 10.294 2L 5 grams and 6 cycles no 10.875 2L 5 grams and 6 cycles no 19.826 2L 5 grams and 6 cycles no 26.3Lysate from kit - - - 30.32

Table 4: Protein concentration for the 6 different lysates. The last entry corresponds to the proteinconcentration from the lysate of Invitrogen cell-free kit.

was already observed in other studies [7]. Finally,the volume of lysate added was tested (6c). Sincethe lysate buffer has a lower protein concentration,increase lysate volume added should increase theprotein produced. This seems to be observed.

5. Conclusions

Although the main goal of construct a reproducibleE. coli cell-free protein synthesis system platformwas not achieved, some major step were taken inthis path. In the future, it would be importantto improve the lysate production. Different lysismechanism (such as high pressure homogeniser ora sonicator) should be tested. Also, lowering thecentrifugation speed and produce the cell culturewith pH control could affect results. Another possi-ble research direction could be to test other energysystems, such as dual energy system and maltoseenergy system. Finally, it seems that the configu-ration also affects protein production: it would im-portant to experiment with a bilayer system or athin film approach.

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(a)

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Figure 6: Graphics with the optimisation screensof E. coli cell-free protein synthesis contianing thecytomimic system as a energy system, pEXP5-NT/GFP as a template and the lysate 6. 6a) Ac-tive GFP produced, in relative fluorescence units,with the variation of magnesium concentration from2mM to 12mM in increments of 2mM. 6b) ActiveGPF produced, in relative fluorescence units, withthe variation of template (pEXP5-NT/GFP) con-centration in µg×µL−1. 6c) Active GPF produced,in relative fluorescence units, with the variation oflysate volume (%).

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