silva et al-2015-journal of separation science

www.jss-journal.com

JOURNAL OF

SEPARATIONSCIENCE

ISSN 1615-9306 · JSSCCJ 38 (5) 703–882 (2015) · Vol. 38 · No. 5 · March 2015 · D 10609

MethodsChromatography · Electroseparation

ApplicationsBiomedicine · Foods · Environment

732 J. Sep. Sci. 2015, 38, 732–740

Marta S. Silva1

Vania C. Graca2

Lucinda V. Reis2

Paulo F. Santos2

Samuel Silvestre1

Luiza Granadeiro1

Paulo Almeida1

Joao A. Queiroz1

Fani Sousa1

1CICS-UBI—Centro deInvestigacao em Ciencias daSaude, Universidade da BeiraInterior, Covilha, Portugal

2Dept. Quımica and Centro deQuımica—Vila Real,Universidade de Tras-os-Montese Alto Douro, Vila Real, Portugal

Received June 14, 2014Revised December 7, 2014Accepted December 18, 2014

Research Article

3,3′-Diamino-N-methyldipropylamine as aversatile affinity ligand

Currently, in biomedicine and biotechnology fields, there is a growing need to develop andproduce biomolecules with a high degree of purity. To accomplish this goal, new purifica-tion methods are being developed looking for higher performance, efficiency, selectivity,and cost-effectiveness. Affinity chromatography is considered one of the most highly se-lective methods for biomolecules purification. The purpose of this work is to explore anew type of a structurally simple ligand immobilized onto an agarose matrix to be usedin affinity chromatography. The ligand in this study, 3,3′-diamino-N-methyldipropylaminehas shown low toxicity and low cost of preparation. Moreover, the ability of the ligand tobe used in affinity chromatography to purify proteins and nucleic acids was verified. Anincreasing sodium chloride gradient, using salt concentrations up to 500 mM, was suit-able to accomplish the purification of these biomolecules, meaning that the new supportallows the recovery of target biomolecules under mild conditions. Thus, the 3,3′-diamino-N-methyldipropylamine ligand is shown to be a useful and versatile tool in chromatographicexperiments, with very good results either for proteins or supercoiled plasmid isoformpurification.

Keywords: Affinity chromatography / 3,3′-Diamino-N-methyldipropylamineligand / Supercoiled plasmid DNADOI 10.1002/jssc.201400656

1 Introduction

In the biotechnology industry the capacity to producesubstantial quantities of safe, pure, and active therapeuticbiomolecules is an open-ended challenge. New technologiesare being pursued in this field to achieve high throughput,cost-effective, and flexible manufacturing processes [1–5].In what concerns the purification methodologies, affinitychromatography is a widely used method to reach the highpurity level required for biopharmaceutical products. In fact,this chromatographic method is considered the most selec-tive for biomolecules purification [1, 2, 6–8], enabling us toovercome some limitations associated with other chromato-graphic techniques, namely size-exclusion, anion-exchange,reversed-phase, or hydrophobic interaction chromatography.The lack of selectivity to separate biomolecules with identicalsize and the high sample dilution are the main drawbacksof size-exclusion chromatography [8, 9]. In addition, thereare some reports [9, 10] describing the poor selectivity ofanion-exchange chromatography for the purification of

Correspondence: Dr. Fani Sousa, CICS-UBI—Centro deInvestigacao em Ciencias da Saude, Universidade da BeiraInterior, Av. Infante D. Henrique, Covilha 6200-506, PortugalE-mail: fani.sousa@fcsaude.ubi.ptFax: +351-275-329099

Abbreviations: DAMDPA, 3,3′-diamino-N-methyldipropy-lamine; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; pDNA, plasmid DNA

nucleic acids and short proteins. Reversed-phase chromatog-raphy must be avoided in the purification of biomoleculeswith biomedical application, because the organic solventsused in the chromatographic process can damage thebiomolecules structure [9]. On the other hand, hydrophobicinteraction chromatography can also present some disad-vantages because of the limited specificity to purify slightlyhydrophobic biomolecules and the need to use high saltconcentrations [11]. According to several authors [12–14],the most powerful chromatographic technique is affinitychromatography, which is rapid, highly selective, and allowsthe recovery of biomolecules maintaining their biologicalactivity.

Conventional affinity ligands such as peptides, oligonu-cleotides, antibodies, and binding or receptor proteins [7] areextremely specific in most cases but can also be expensiveand difficult to immobilize preserving their biological activ-ity [6]. Synthetic ligands may circumvent these drawbacksbecause they are relatively inexpensive, easily immobilizedin matrices and resistant to chemical and biological degra-dation [2, 15]. Dye ligands for affinity chromatography havebeen mainly based on triazine dyes, both the conventionalones, picked from the textile industry, and the syntheticbiomimetic dyes, tailored specifically for the isolation of a tar-get biomolecule [16]. Although being one of the most popularclass or organic dyes, cyanine dyes have been scarcely usedas ligands. Recently, we reported the use of several symmet-rical aminosquarylium cyanine dyes derived from benzothia-zole as ligands for the separation of a mixture of lysozyme,

J. Sep. Sci. 2015, 38, 732–740 Liquid Chromatography 733

�-chymotrypsin, and trypsin [17]. In this case, the dye was im-mobilized in Sepharose using diaminoalkyl groups as spacerarms.

Moreover, affinity chromatography with amino acids asligands has recently yielded interesting results to purify plas-mid DNA [18], by promoting specific interactions with dif-ferent nucleic acids [9]. Histidine, arginine, and lysine thatbelong to the positively charged amino acids group, can beused as affinity ligands and allow successful purification ofplasmid DNA (pDNA) from complex E. coli lysates, show-ing a specific biorecognition toward the supercoiled pDNAconformation.

Following our interest in the development of new affinitychromatographic supports for the separation and purificationof biomolecules using squarylium dyes as ligands, we havebeen exploiting several different aminoalkyl spacer arms forsquarylium dyes envisioning the increase of the spatial mo-bility of the immobilized ligand and, consequently, of thedye–biomolecule interaction and binding capacity of the sup-port. Unexpectedly, we ended with a very efficient nondyedchromatographic matrix for the separation of proteins andpDNA, consisting of Sepharose functionalized with a simpletriamine, 3,3′-diamino-N-methyldipropylamine (DAMDPA).In the present work Sepharose, a cross-linked, beaded-formof agarose, was used because it is chemically and phys-ically more resistant and presents better flow propertiesthan the common Agarose. To prove the versatility of thenew support, it was used to purify different biomolecules,such as standard proteins and pDNA. The choice of thesebiomolecules will bring important insights concerning therobustness of this new ligand to interact either with proteinsor nucleic acids, allowing the potential application to theirpurification.

2 Materials and methods

2.1 Materials

All reagents used in the preparation of the chromato-graphic support and BSA (from bovine serum), k-casein(from bovine milk), and Ribonuclease A (RNase A, frombovine pancreas) were purchased from Sigma–Aldrich(St. Louis, MO, USA). Solvents were of analytical grade.For chromatographic assays, sodium chloride was purchasedfrom Panreac (Barcelona, Spain) and Tris base from FisherScientific (Leicestershire, UK). All solutions were freshly pre-pared using ultra-pure grade water, purified with a milli-Qsystem from Millipore (Billerica, MA, USA) and filtered us-ing a 0.20 �m pore size membrane (Schleicher Schuell, Das-sel, Germany). Qiagen Plasmid Purification Maxi Kit wasacquired from Qiagen (Hilden, Germany). Low-molecular-weight protein marker from GE Healthcare (Sweden) wasused in SDS-PAGE and the DNA molecular weight marker,hyper ladder II, was obtained from Bioline (London).

2.2 Preparation of 3,3′-diamino-N-

methyldipropylamine-Sepharose support

2.2.1 Activation of Sepharose CL-6B

A suspension of Sepharose CL-6B in EtOH (50 mL of set-tled gel) was placed into a sintered glass funnel and care-fully washed thoroughly with water, under reduced pressure.The gel was placed in an Erlenmeyer flask and suspendedin 50 mL of 0.2 M aqueous NaIO4. The mixture was keptunder gentle orbital agitation, at room temperature, for 1 h.The activated support was then collected by filtration underreduced pressure and washed with water (500 mL). The acti-vated Sepharose was stored in 0.02% aqueous NaN3 (100 mL)and kept refrigerated.

2.2.2 Coupling of 3,3′-diamino-N-

methyldipropylamine to activated Sepharose CL-6B

To a solution of DAMDPA (12.3 mL, 76.13 mmol) in water(25 mL), cooled in an ice bath, was added concentrated HClunder vigorous stirring until pH 7. Solid Na3PO4 was thenadded to obtain a concentration of 0.1 M (based on the finalvolume of solution) (2.10 g, 5.55 mmol). The solution pHwas again adjusted to 7 with concentrated HCl. Then, NaIO4-activated Sepharose CL-6B (50 mL of settled gel) washed con-secutively, under reduced pressure, with water and 0.1 MNa3PO4 buffer (pH 7), was transferred to the above solu-tion of DAMDPA, followed by the addition of NaCNBH3

(0.60 g, 9.55 mmol). The mixture was kept under gentle or-bital agitation, at room temperature, for 4 h. The gel was thencollected by filtration under reduced pressure and washed,sequentially, with water (1 L), 1 M aqueous NaCl (500 mL),water (500 mL), and 0.2 M aqueous AcONa. Then, to an ice-cooled suspension of the washed gel in 0.2 M aqueous AcONa(10 mL), under orbital agitation, was added cold Ac2O (5 mL).After 30 min more Ac2O (5 mL) was added and the mixturekept at room temperature for an additional identical period.The gel was then placed into a sintered glass funnel andwashed thoroughly with water and acetone, under reducedpressure [19]. Finally, the gel (Fig. 1) was suspended in ace-tone and kept in the refrigerator.

2.3 Bead morphology

Concerning the preparation of a new chromatographic sup-port, it is important to characterize the bead morphology toguarantee that the immobilization process is not affecting theproperties of the material. With this purpose, the beads mor-phology was evaluated, before and after the immobilizationprocess, using SEM (Hitachi S-2700, Tokyo, Japan), operatedat an accelerating voltage of 20 kV, at variable magnifications.The samples were fixed on a brass stub using double-sidedtape and then made electrically conductive by coating withgold using an Emitech K550 sputter coater (London).

734 M. S. Silva et al. J. Sep. Sci. 2015, 38, 732–740

Figure 1. Preparation of DAMDPA-activated Sepharose CL-6B by periodate oxidative cleavage of vicinal diols present at sites of incompletecross-links on the gel, followed by reductive amination of the aldehyde groups generated [19].

2.4 Toxicity assays

The leakage of ligand is a matter that should be considered insynthetic-based affinity supports because minimum amountsof ligands can be lost during the chromatographic process.For this assay, human breast cancer cells (MCF-7; acquiredfrom the American Type Culture Collection) were used.Chemicals (analytical grade), assay reagents, culture media,and supplements were all acquired from Sigma–Aldrich.

2.4.1 Culture of cells

Cells were routinely maintained at 37�C in a humidified at-mosphere containing 5% CO2. Dubelco’s Modified Eagle’sMedium high glucose, containing phenol red, and supple-mented with 10% fetal bovine serum (FBS) and 1% antibi-otic/antimycotic, was used to culture MCF-7 cells. The cellswere used in passages nine and ten in the experiments.

2.4.2 Analysis of cell viability

Cell viability was studied quantifying the extent of the reduc-tion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) according to a previously described proce-dure [20]. Briefly, cells were seeded in 48-well plates (0.5 ×104 cells/well) in the culture medium containing FBS andafter 48 h they were treated with six concentrations (7500,750, 75, 7.5, 0.75, and 0.075 �g/mL) of the ligand and themodified support in the complete culture medium for 48 h,with untreated cells serving as control. At the end of incu-bation the media in wells were removed, replaced with freshmedia and MTT solution and incubated at 37�C for 4 h. There-after, media-containing MTT were removed, formazan crys-tals were dissolved and absorbance was recorded in an Anthos2020 microplate reader at 570 nm. The extent of cell deathwas expressed as the percentage of cell viability in comparisonwith control cells.

2.4.3 Statistical analysis

The cell viability experiments were performed in quintupli-cate and the graphical results of cell proliferation were ex-pressed as average ± SD. The comparison between groupswas analyzed using Student’s t-test and the differences be-tween groups were considered statistically significant atp < 0.05. The half-medium inhibitory concentration (IC50)value was calculated from the obtained dose–response curveby sigmoid fitting.

2.5 Bacterial growth conditions and plasmid

isolation

The 6.05kbp plasmid pVAX1-LacZ (Invitrogen, Carlsbad, CA,USA) was produced by a cell culture of E. coli DH5�. Growthwas carried out at 37�C using Terrific Broth medium (24 g/Lof yeast extract, 20 g/L of tryptone, 4 mL/L of glycerol, 0.017 MKH2PO4 and 0.072 M K2HPO4) supplemented with 30 �g/mLof kanamycin. Cells were recovered by centrifugation at thelate log phase (OD600 � 6) and stored at –20�C. Plasmid DNAwas isolated and prepurified using the Qiagen Plasmid MaxiKit, according to the manufacturer’s instructions. The proto-col is based on a modified alkaline lysis procedure. Followinglysis, binding of pDNA to the Qiagen anion-exchange resin ispromoted under appropriate low-salt and pH conditions. Im-purities are removed by a medium-salt wash. Plasmid DNAis eluted in a high salt buffer and then concentrated and de-salted by isopropanol precipitation. The prepurified pDNAsample contains both supercoiled and open circular isoformsand was used to evaluate the ability of the DAMDPA-basedsupport to isolate plasmid isoforms.

2.6 Chromatographic method

In a 16 mm (diameter) × 200 mm (length) column, approx-imately 6 mL of the prepared gel was packed. The chromato-graphic runs were performed in an AKTATM purifier system

with UNICORN software, version 5.11 (GE HealthcareBiosciences, Uppsala, Sweden). The experiments wereconducted at a flow rate of 1 mL/min and a water jacketconnected to a circulating water bath was used to maintainthe temperature around 10�C. The experiments developedfor the purification of proteins were initiated with the equili-bration of the column with 10 mM Tris–HCl buffer, pH 7.0.The sample was loaded onto the column using a 200 �L loopand the absorbance was measured at 280 nm. The elution ofunbound proteins was achieved with 10 mM Tris–HCl, pH7.0 and the recovery of retained proteins was carried out byincreasing the NaCl concentration to 500 mM NaCl in 10 mMTris–HCl, pH 7.0, using a stepwise gradient. Similarly, theexperiments aiming at pDNA purification, started with thecolumn equilibration with 10 mM Tris–HCl buffer, pH 8.0.The pDNA samples were injected onto the column using a200 �L loop and the absorbance was monitored at 260 nm. Forthe elution of unbound species it was used 10 mM Tris–HClbuffer, pH 8.0 and the retained pDNA species were recoveredby increasing the ionic strength of the buffer to 500 mM NaClin 10 mM Tris–HCl, pH 8.0, using a stepwise gradient. Con-sidering the chromatographic profiles obtained for proteinsor pDNA purification, the fractions corresponding to thepeaks were collected, desalted and concentrated for furtheranalysis.

To maintain the performance of this chromatographicsupport, it was always performed an efficient washingstep with 1 M of NaCl, between runs. With this proce-dure it was verified the reproducibility for more than 50experiments.

2.7 SDS–PAGE

The purity of the protein fractions recovered from the chro-matographic runs was evaluated by 15% SDS-PAGE using aBio-Rad system (Bio-Rad, Hercules, CA, USA) according tothe manufacturer’s protocol. Briefly, protein samples weredenatured by the addition of loading dye (62.5 mM Tris-HCl,pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue)followed by incubation at 100�C for 10 min. Electrophoresiswas carried out at 150 V for 90 min with Tris/glycine/SDSbuffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3) and the gel was stained by Coomassie Brilliant BlueR-250.

2.8 Agarose gel electrophoresis

The pDNA fractions recovered from each chromatographicexperiment were analyzed using 1% agarose gels (Hoefer,San Francisco, CA, USA), dyed with GreenSafe direct load(1 �g/mL). Electrophoresis was carried out at 110 V, for35 min, with TAE buffer (40 mM Tris base, 20 mM aceticacid, and 1 mM EDTA, pH 8.0). The gel was visualized un-der UV light in a Vilber Lourmat system (ILC Lda, Lisbon,Portugal).

3 Results and discussion

3.1 Characterization of 3,3′-diamino-N-

methyldipropylamine-based support

The preparation and evaluation of a new chromatographicsupport (Fig. 1) requires a complete characterization ofthe ligand and the support to confirm its applicability inbiomolecules purification. Therefore, the ligand density ofthe support, the beads morphology as well as the toxicity as-sociated to the ligand, were evaluated since the potential ap-plication of the chromatographic support may be constrainedby these parameters.

The ligand density of the support was found to be 0.395mmol per gram of support and was estimated from thepercentage of nitrogen determined by elemental analysis.This value is slightly higher than the ligand density of otherrecently prepared affinity matrices [17] using benzothiazole-based squarylium dyes as ligands, what can be advantageousfor the binding of biomolecules and the capacity of thesupport.

The characterization of the morphology of Sepharosebeads prior and after immobilization was also performedby SEM. Figure 2 illustrates beads morphology after theimmobilization of the DAMDPA ligand (A–D) in compari-son with the morphology of the nonimmobilized Sepharose(1–4). In fact, no significant differences could be observed,suggesting that the immobilization procedure did not causevisible damages in the support. With this result, it is ex-pected that the chromatographic performance of the sup-port regarding the flow properties is maintained, and thatthe separation ability should be only influenced by theligand.

To accomplish the global characterization of the sup-port, it was also evaluated the toxicity of the ligand. Someresidual leakage can occur when synthetic ligands are be-ing used. However, it is crucial to understand the impactof the leakage in the purified samples. The toxicity studieswere performed using MCF-7 cells that were exposed for48 h to six different concentrations (7500, 750, 75, 7.5, 0.75,and 0.075 �g/mL) of either the ligand (DAMDPA) or themodified support, using untreated cells as control. Accord-ing to the data presented in Fig. 3, it is apparent that thechromatographic support obtained after immobilization ofDAMDPA has low toxicity to the MCF-7 cells under theseconditions. In fact, only a small decrease in cell viability wasobserved for the two highest concentrations of the support, incomparison with the control. These results clearly indicatedthat the support seems to be safe. The isolated ligand itself,however, revealed to be more toxic (IC50 = 0.2542 mg/mL; r2

= 0.9749; 95% confidence interval) than the immobilized sup-port, with a clear decrease in cell proliferation being observedfor the two higher concentrations (750 and 7500 �g/mL). In-terestingly, these last results can be explained by the changein the solutions pH for those two concentrations of DAMDPAin the culture medium. In fact, due to the presence of phe-nol red, we observed a color change from pink to fuchsia in

Figure 2. Scanning electron microscope images of beaded Sepharose CL-6B with 100, 250, 500, and 3000× magnification: (1–4) beforeimmobilization; (A–D) after immobilization.

these two solutions, in comparison with the culture mediumalone, which indicated a pH clearly higher than the physi-ological one (7.4). As cell functionality depends on the pHof the medium, this change is probably responsible for themarked decrease in cell proliferation observed with the higherconcentrations of this basic compound. In spite of the an-tiproliferative activity associated with this compound, it isimportant to mention that these two solutions of DAMDPAhave very high concentrations (750 and 7500 �g/mL),which could only be associated with an unusual and strongchange in the functionalized support, not related with resid-ual leakage. According to the literature [21, 22], when thesupport is submitted to harsh conditions the highest leakageis approximately 1–2 �g/mL, pointing to a minimum risk oftoxicity when using the chromatographic support describedherein.

3.2 Purification of proteins using 3,3′-diamino-N-

To evaluate the ability of the new support to interact and iso-late proteins from a mixture, three standard proteins, namelyBSA, RNase A, and k-casein were used. The protein solu-tions were prepared with a concentration of 15 mg/mL anddissolved in 10 mM Tris-HCl buffer, pH 7.0. The sample(200 �L) was loaded onto the column and the elution was per-formed by increasing the NaCl concentration, using a step-wise gradient. The screening experiments were performedusing isolated proteins, to determine the interaction and rel-ative strength of interaction of each protein with the support.In these assays, different concentrations of NaCl were testedto achieve the most suitable binding and elution conditions(results not shown). After the optimization of the chromato-graphic conditions, an artificial mixture was prepared withthe three proteins. The best conditions were achieved with the

application of a stepwise gradient, where the ionic strengthof the buffer was increased from 0 M to 100 mM and, finally,500 mM NaCl in 10 mM Tris–HCl, pH 7.0. As visible in Fig.4A, the application of the protein mixture onto the columnresulted in a differential interaction, which allowed their elu-tion in three different peaks. The SDS-PAGE analysis (Fig.4B) of the fractions recovered from each peak revealed whichproteins were eluted in the three elution steps. In the bindingstep, it was obtained a first peak corresponding to the elutionof the unbound proteins. As confirmed by SDS-PAGE (lane1), RNase A was not able to interact with the DAMDPA ligand.The second peak (lane 2 of the electrophoresis), correspondsto the recovery of BSA, which is eluted as consequence of anincrease of ionic strength to 100 mM NaCl in 10 mM Tris-HCl buffer, pH 7.0. In the last step, all the interactions wereweakened and, as consequence, the more retained proteinwas eluted. In this case, k-casein was the protein showing astronger interaction with the DAMDPA-based support, as canbe observed in Fig. 4B (lane 3). The identification of the pro-teins eluting in each chromatographic peak was based on thescreening experiments using isolated proteins and on a com-parison of their electrophoretic profile with that of standardpure proteins (lanes A, B, and C).

Concerning the interactions established between theDAMDPA ligand and the three proteins, it is undoubted theinvolvement of electrostatic interactions, due to the positivecharacter of the ligand, given by the amine groups. Moreover,as the experiments were performed at pH 7.0, the RNaseA protein (pI of 9.6) was positively charged, whereas BSAand k-casein presented a negative charge. Considering this,it is easily explained the retention of BSA and k-casein andnonretention of RNase A. However, it was also found a sig-nificant difference on the interaction strength of BSA andk-casein with the ligand, despite the similarity of both pIs.BSA presents an pI of 4.7 and for k-casein there are tenknown forms with pI values from 4.47 to 5.81. In this case,

Figure 3. Relative cell viability ofMCF-7 cells when exposed for 48 hto (A) functionalized Sepharose or(B) DAMDPA, in concentrations rang-ing from 0.075 to 7.5 mg/mL (MTTassay). Data are expressed as a rel-ative % of cell viability in comparisonwith the control; the bars representthe mean and the lines represent theassociated SD; *p < 0.05 versus thecontrol (Student’s t-test, n = 5).

at pH 7.0, it is likely that k-casein presents almost the sameor even lower negative charge than BSA, but it was veri-fied that k-casein suffers a stronger retention on the sup-port. This result indicates that the interaction of proteinswith the ligand is not exclusively due to ionic effects butto multiple noncovalent interactions and thus the supportis not for a typical ion-exchange chromatography. In thelight of these results, it is proposed that the proteins separa-tion is based on multiple noncovalent forces involved in theaffinity interactions established between the proteins and thesupport. Thus, the biomolecular interactions established aremainly due to ionic interactions but other elementary inter-actions are also promoted, namely van der Waals forces and

hydrogen bonds. The involvement of these multiple inter-actions may be responsible for the specificity to distinguishproteins with similar charge characteristics, enabling theirefficient separation.

3.3 Purification of pDNA using 3,3′-diamino-N-

The versatility of a chromatographic support is related tothe ability to be applied on the purification of differentsamples. So, some preliminary assays were also carried outto determine the possibility of using the DAMDPA-based

Figure 4. (A) Chromatographic profile af-ter loading a mixture of k-casein, BSA andRNase A (15 mg/mL), using 10 mM Tris-HCl (pH 7.0) in the initial washing step,followed by a stepwise gradient of NaClfrom 0M to 500 mM (as represented bythe arrows.). (B) SDS-PAGE analysis of thefractions eluted from the chromatographicsupport. Lane 1, RNase A (first peak); lane2, BSA (second peak); lane 3, k-casein(third peak); lane M, artificial mixture ofthe three proteins; lane A, RNase A (con-trol); lane B, BSA (control); lane C, k-casein(control).

support to purify plasmid DNA samples. Native plasmid sam-ples, containing both supercoiled (sc) and open circular (oc)isoforms, were loaded onto the column using a 200 �L loopand the elution was tested by increasing the ionic strengththrough a stepwise gradient with NaCl. The global aim was toverify the ability of the DAMDPA ligand to isolate both plas-mid isoforms, to purify the sc pDNA conformation, which isendowed with higher biological activity.

In the binding step, it was used 10 mM Tris-HCl buffer,pH 8.0 and both isoforms were retained. After a total reten-tion of pDNA, a selective elution of oc and sc pDNA isoformswas achieved as observed in the chromatographic profile andconfirmed by agarose gel electrophoresis (Fig. 5). The ionicstrength of the buffer was increased to 130 mM NaCl in10 mM Tris-HCl buffer, pH 8.0. In this step, the weak inter-actions established are broken and starts the elution of the oc

isoform (peak 1). Subsequently, the concentration of salt wasincreased to 500 mM NaCl in 10 mM Tris-HCl buffer, pH 8.0,promoting the elution of the second peak which correspondsto the sc pDNA isoform. The purity of these molecules wasattested by agarose gel electrophoresis (Fig. 5). In this figure,we can see in lane S the sample injected in the column con-taining both plasmid isoforms, in lane 1 the oc isoform elutedin peak 1 and the sc isoform in lane 2, which was recoveredfrom peak 2 of the chromatographic experiment.

The sc pDNA isoform elutes in the last step, as con-sequence of an increase in the ionic strength. This resultshows that the strongest interaction with the support wasestablished by the sc pDNA isoform and also indicates thatthe different pDNA isoforms interact differently with thesupport, allowing their purification. Plasmid DNA isoformspurification was achieved due to the affinity interactions

Figure 5. Chromatographicprofile showing the effect ofthe ionic strength (representedby the dashed line and by thearrows) on the separation ofpDNA isoforms. Agarose gelelectrophoretic analysis ofsamples recovered in the chro-matographic process. LaneM, DNA molecular weightmarker; lane S, pDNA sampleinjected onto the column (oc+ sc); lane 1, oc isoform (peak1); lane 2, sc isoform (peak 2).

established between this biomolecule and the support.Despite the polyanionic character of the pDNA molecule,owing to the presence of phosphate groups on the nucleicacid backbone, the electrostatic interactions are not the onlyforces involved in the biorecognition of the sc pDNA isoform.The negative charge of pDNA promotes the retention of bothisoforms in the column, but the specific interaction with thesc pDNA is related to the different exposure of the nucleotidesbases in sc and oc pDNA isoforms [23]. The supercoilingphenomenon promotes a higher exposure of the sc pDNAbases [24], enabling their access to the ligands and promotinga stronger interaction, when compared with the oc conforma-tion. These results are in accordance with what was previouslydescribed for arginine-affinity chromatography [23, 25]. Inthat case, the multiple interactions that the arginine-based matrix is able to promote, allowed the differentialrecognition of the biomolecules present in E. coli lysates,enabling the purification of the sc pDNA under mild elutionconditions.

Overall, as previously discussed for the proteins purifica-tion, the DAMDPA ligand enabled the purification of pDNAbased on the multiple noncovalent interactions that occurred,namely, electrostatic, hydrogen bonds, and van der Waalsforces. Besides the specificity to recognize the sc plasmidDNA isoform, we also confirmed the versatility of the ligandto purify either proteins or plasmids. The majority of the chro-matographic studies of new ligands are focused on the purifi-cation of a target biomolecule. For example, arginine-affinity

chromatography was successfully used by Sousa and Queiroz,and Martins et al. to purify DNA and RNA, respectively[26,27]. In 2010, Grodzki and Berenstein, described a methodfor the purification of immunoglobulins (IgG, IgG fragments,and subclasses) using the high affinity of protein A and pro-tein G coupled to agarose [28]. An Other powerful purificationtechnique, based on immobilized metal-affinity chromatog-raphy was also used by Bornhorst and Falke to purify recom-binant proteins [29]. In the present study, it was verified thatthe ligand herein described can be used to purify two differ-ent types of biomolecules, which may represent an advantageconcerning the robustness of the chromatographic supports.

4 Concluding remarks

To overcome a lack of suitable supports that can be usedwith different types of biomolecules, some efforts were madeto study and create a versatile ligand. The main goal of thiswork was successfully achieved because it was possible toapply DAMDPA as a chromatographic ligand to efficientlypurify either proteins or nucleic acids. The morphology ofSepharose beads was studied before and after immobiliza-tion, and it was concluded that no changes occurred in thestructure of beads after immobilization of the ligand. More-over, the toxicity experiments revealed that it is possible tosafely use the chromatographic support. Globally, the resultsindicate that a versatile affinity ligand is being used in this

work, since it was applied to separate three different standardproteins and it was also possible to use it to efficiently separatepDNA isoforms. Moreover, in this methodology mild condi-tions were used, without high salt concentrations, which isan advantage in the point of view of establishing an upgradein downstream strategies for industrial applications reducingthe costs and environmental impact.

This work was supported by the Portuguese Foundationfor Science and Technology, Portugal, POCTI and FEDER,by the Project “PTDC/QUI-QUI/100896/2008” and PEst-C/SAU/UI0709/2011 COMPETE. M. S. Silva and V. C. Gracaalso acknowledge fellowships in the ambit of this project. We arealso grateful to the Optical Center of University of Beira Interiorfor the acquisition of the SEM images.

The authors have declared no conflict of interest.

5 References

[1] Clonis, Y. D., Labrou, N. E., Kotsira, V. P., Mazitsos, C.,Melissis, S., Gogolas, G., J. Chromatogr. A 2000, 891,33–44.

[2] Sproule, K., Morrill, P., Pearson, J. C., Burton, S. J.,Hejnæs, K. R., Valore, H., Ludvigsen, S., Lowe, C. R., J.Chromatogr. B Biomed. Sci. Appl. 2000, 740, 17–33.

[3] Sousa, A., Sousa, F., Queiroz, J. A., J. Sep. Sci. 2012, 35,3046–3058.

[4] Schiel, J. E., Mallik, R., Soman, S., Joseph, K. S., Hage,D. S., J. Sep. Sci. 2006, 29, 719–737.

[5] Roberts, M. W. H., Ongkudon, C. M., Forde, G. M., Dan-quah, M. K., J. Sep. Sci. 2009, 32, 2485–2494.

[6] Clonis, Y. D., J. Chromatogr. A 2006, 1101, 1–24.

[7] Lowe, C. R., Lowe, A. R., Gupta, G., J. Biochem. Biophys.Methods 2001, 49, 561–574.

[8] Stulık, K., Pacakova, V., Ticha, M., J. Biochem. Biophys.Methods 2003, 56, 1–13.

[9] Sousa, A., Sousa, F., Queiroz, J. A., J. Sep. Sci. 2010, 33,2610–2618.

[10] Tan, S. C., Yiap, B. C., J. Biomed. Biotechnol. 2009, 2009,574398.

[11] Sousa, F., Freitas, S., Azzoni, A. R., Prazeres, D. M. F.,Queiroz, J., Biotechnol. Appl. Biochem. 2006, 45, 131–140.

[12] Hage, D. S., Clin. Chem. 1999, 45, 593–615.

[13] Urh, M., Simpson, D., Zhao, K., Methods Enzymol. 2009,463, 417–438.

[14] Fassina, G., Ruvo, M., Palombo, G., Verdoliva, A.,Marino, M., J. Biochem. Biophys. Methods 2001, 49, 481–490.

[15] Ye, L., Xu, A., Cheng, C., Zhang, L., Huo, C., Huang, F.,Xu, H., Li, R., J. Sep. Sci. 2011, 34, 3145–3150.

[16] Labrou, N. E., Mazitsos, K., Clonis, Y. D., in: Hage, D. S.(Ed.), Handbook of Affinity Chromatography, 2nd Edi-tion, CRC Press, Boca Raton, 2006, pp. 231–255.

[17] Silva, M. S., Graca, V. C., Reis, L. V, Santos, P. F., Almeida,P., Queiroz, J. A., Sousa, F., Biomed. Chromatogr. 2013,27, 1671–1679.

[18] Sousa, F., Prazeres, D. M. F., Queiroz, J. A., TrendsBiotechnol. 2008, 26, 518–525.

[19] Stults, N. L., Asta, L. M., Lee, Y. C., Anal. Biochem. 1989,180, 114–119.

[20] Freshney, R. I., Culture of Animal Cells: A Manual of Ba-sic Technique and Specialized Applications, 6th Edition,Wiley-Blackwell, Hoboken 2010.

[21] Santambien, P., Sdiqui, S., Hubert, E., Girot, P., Roche,A. C., Monsigny, M., Boschetti, E., J. Chromatogr. B.Biomed. Appl. 1995, 664, 241–246.

[22] Thillaivinayagalingam, P., Newcombe, A. R., O’Donovan,K., Francis, R., Keshavarz-Moore, E., Biotechnol. Appl.Biochem. 2007, 48, 179–188.

[23] Sousa, F., Prazeres, D. M. F., Queiroz, J. A., Arch. Biochem.Biophys. 2007, 467, 154–162.

[24] Sousa, F., Matos, T., Prazeres, D. M. F., Queiroz, J. A.,Anal. Biochem. 2008, 374, 432–434.

[25] Sousa, F., Prazeres, D. M. F., Queiroz, J. A., J. Gene Med.2009, 11, 79–88.

[26] Sousa, F., Queiroz, J. A., J. Chromatogr. A 2011, 1218,124–129.

[27] Martins, R., Queiroz, J. A., Sousa, F., Anal. Bioanal. Chem.2013, 405, 8849–8858.

[28] Grodzki, A. C., Berenstein, E., Methods Mol. Biol. 2010,588, 33–41.

[29] Bornhorst, J. A., Falke, J. J., Methods Enzymol. 2000,326, 245–254.

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