Journal of Membrane Science 280 (2006) 298–310

Recognition based separation of HIV-Tat protein using avidin–biotininteraction in modified microfiltration membranes

Saurav Datta a, Philip D. Ray b, A. Nath c, D. Bhattacharyya a,∗a Department of Chemical & Materials Engineering, University of Kentucky, Lexington, KY 40506-0046, United States

b Department of Anatomy and Neurobiology, University of Kentucky, United Statesc Departments of Neurology and Neuroscience, Johns Hopkins University, United States

Received 13 October 2005; received in revised form 13 January 2006; accepted 16 January 2006Available online 20 February 2006

Abstract

Recognition based separation using modified microfiltration membranes provides an efficient and cost-effective alternative to conventionalcolumn chromatography for the separation and purification of a specific protein from mixture of proteins. In this study, Tat protein, which hasbeen proposed as the specific target for AIDS vaccine, was separated and purified from a complex mixture of proteins, known as bacterial lysate(mitbm©

K

1

bpiCusfihc

mkTo

0d

BL) using avidin–biotin interaction in 4-stack microfiltration membranes system. It was established by SDS-PAGE and Western Blot analysis thatembrane based process recovered more pure form of Tat compared to conventional packed-bead column chromatography. The critical factors

nvolved in the process, mainly, the accessibility of the covalently immobilized avidin sites by the biotinylated protein and the associated fouling ofhe membranes due to the permeation of proteins, were also studied. The accessibility of immobilized avidin sites in membrane was quantified byiotinylated solutions of different types and compositions. It was observed that permeation of proteins caused substantial fouling on the membraneatrix. The resistance offered by the protein layer and the approximate thickness of the protein layer were also quantified.2006 Elsevier B.V. All rights reserved.

eywords: Tat; Avidin; Biotin; Accessibility; Fouling

. Introduction

Downstream processing of proteins is very important iniotechnological and pharmaceutical industries. Separation andurification of a protein from mixture require high selectivity andncur a major part of the total production cost of the protein [1].onventional packed-bead column chromatography is widelysed for this type of separation [2]. However, membrane basedeparation processes involving microfiltration (MF) and ultra-ltration (UF) membranes are emerging as cost effective andydrodynamically favorable alternative to conventional columnhromatography [1,3–9].

Recognition based separation of a specific protein fromixture of proteins in MF membranes is achieved using the

nowledge of molecular biology and membrane technology.he technique is also known as membrane chromatographyr affinity membrane separation. It consists of the following

∗ Corresponding author. Tel.: +1 859 2572794; fax: +1 859 3231929.E-mail address: db@engr.uky.edu (D. Bhattacharyya).

steps: (1) immobilization of a ligand that has specific affinitytowards the target protein on the pore walls of MF membrane,(2) selective attachment of the target protein from a mixtureto the membrane by ligand–protein interaction, (3) purifica-tion of the attached protein and elution from the membrane bydisrupting protein–ligand interaction. The activity of the immo-bilized ligand depends on the method of immobilization in themembranes. It can be done either by random covalent immo-bilization or by site-specific immobilization with the help ofamine, aldehyde, epoxide, or anhydride group already presentin the membrane matrix [10]. MF membranes allow the pro-tein molecules to permeate through the pores, and convectiveflow helps them to access the immobilized ligand easily. Some-time a tag is introduced in the target protein to separate itusing ligand–tag interaction [4]. The commonly used interac-tions are avidin–biotin, antigen–antibody, enzyme–substrate, oroligonucleotide–protein binding [8].

On the other hand, in UF, proteins are separated basedon their molecular weight, using particular molecular-weight-cut-off (MWCO) membranes. Sometimes, moderately differentmolecular weight proteins are also separated by manipulating

376-7388/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.memsci.2006.01.031

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 299

the operating conditions [11–13]. Stacked ultrafiltration mem-branes have also been used to separate proteins closer in sizefrom mixture with higher selectivity [14].

The feasibility of UF and MF processes is limited byprotein–membrane and protein–protein interactions that lead todecrease in the permeate flux with time due to irreversible alter-ation or fouling of the membrane surface and pore structure[15–24]. Fouling depends on feed properties, membrane mate-rials and operating conditions. In general, the flux decline dueto membrane fouling can be described in terms of four blockingmechanisms [17].

Among the aforementioned interactions, avidin–biotin iswidely used because of the high association constant (1015 M−1)[25]. The mechanism of association between avidin and biotin isnot well known. However, it is already established, that 1 moleof tetrameric avidin in ideal situation could bind approximately4 moles of biotin. Avidin–biotin interaction has been used bymany researchers to immobilize enzymes on the membrane ina site-specific manner [10,26–27]. Avidin–biotin interaction inmembrane matrix has also been used for the recognition basedseparation of a protein from mixture [4]. However, the acces-sibility of avidin sites in the membrane matrix has not beenquantified.

TAT (trans-activating transduction) protein is a regulatoryprotein of human immunodeficiency virus type 1 (HIV-1)[28–30]. It is a highly basic protein, and therefore, has highpTslretatori[

trri

autaftos((ag

to the permeation of proteins and protein mixtures. The effectof pretreatment of BL on free available Tat was also studied.

2. Experimental methods

2.1. Equipment and materials

The acylanhydride functionalized nylon based MF mem-branes (Immunodyne ABC; lot numbers: ND1409 and ND0311)used in all the experiments were purchased from Pall Corpora-tion. The pore size of the membranes was 0.45 �m and the thick-ness was 165 �m. Immunodyne membranes are also available in1.2 �m pore size. However, to obtain higher internal surface areaper unit volume and minimize core leakage through the centralregion of the pores, 0.45 �m was selected instead of 1.2 �m.

Stirred membrane cell purchased from Millipore Corporation(Product number XFUF07601) was used for all the experiments.To ensure complete mixing in the cell, mixtures were stirred witha magnetic stirrer. In order to determine that there is no bypassof the membrane stack after it was assembled into the stirredcell, controlled experiments were performed by permeating BSA(molecular weight 67,000) solution through 5000 MWCO PESUF membranes stacked in the stirred cell. Absence of BSA inpermeate has eliminated the possibility of bypass in the cell.

Biotinylated-bovine serum albumin (BBSA), factor Xa andBiotin Quantification Kit were purchased from Pierce Biotech-nfUfpv

2

poN

30m1aitrAhmttasci

otential for non-specific binding with different RNAs [31].he primary role of Tat is to regulate (productive and proces-ive) transcription from the HIV-1 promoter region termed theong terminal repeat (LTR) [32]. Tat not only plays a criticalole in HIV replication but also has a number of deleteriousffects on the immune and nervous systems [33]. This pro-ein is potentially an excellent target for AIDS related vaccinend drug development. However, the purification of Tat pro-ein presents several challenges. It binds to a large numberf cellular proteins and due to the presence of a cysteine richegion tends to form large complex polymers. Tat is functionaln the monomeric form, not in the dimeric or polymeric forms34].

Separation of Tat from bacterial lysate (BL) by recogni-ion based membrane process has been reported earlier by ouresearch group [4]. That research work had proposed that supe-ior quality of Tat can be isolated using membrane separationsn comparisons to column chromatographic separation.

The main objective of this research work was to separatend purify monomeric Tat protein from bacterial lysate (BL)sing avidin–biotin interaction in modified stacked microfiltra-ion membranes. Accessibility of the covalently immobilizedvidin sites in the membranes by the biotinylated protein and theouling of the membranes due to permeation of proteins werewo major concerns associated with this separation. Thus, otherbjectives of this research work were: (1) to quantify the acces-ibility of covalently immobilized avidin sites by permeatinga) a small biotinylated compound, biotin 4-amidobenzoic acidBABA), (b) a biotinylated protein, biotinylated-BSA (BBSA),nd (c) a mixture of BBSA and a non-biotinylated protein,amma globulin (GG). (2) To study the associated fouling due

ology. Gamma globulin (GG) and the reagents for Brad-ord protein assay were purchased from Bio-Rad Laboratories.nless specified otherwise, all other chemicals were purchased

rom Sigma Corporation. The bacterial lysate supernatant wasrepared in the Department of Anatomy and Neurobiology, Uni-ersity of Kentucky.

.2. Permeation of BABA and BBSA

Accessibility of avidin in homogeneous phase by biotinresent in BABA (1 mole of BABA is equivalent to 1 molef biotin) was determined using avidin and BABA in 0.05 Ma2HPO4 buffer.Single membrane (diameter 6.5 cm, external surface area

3.18 cm2, bed volume = external surface area × thickness =.55 ml) was used in the stirred membrane cell for the per-eation of (i) BABA (0.6, 3.2 and 9.6 �g/ml), (ii) BBSA (3,

0, 19.3 �g/ml), and (iii) 10 �g/ml BBSA in a mixture withnon-biotinylated protein, gamma globulin (GG). Avidin was

mmobilized by permeating in 0.05 M Na2HPO4 buffer at roomemperature and 0.68 bar (10 psi) pressure with constant stir-ing at 300 rpm. Pressure in the cell was maintained by N2 gas.vidin molecules became covalently attached with the acylan-ydride groups present in the membrane following the reactionechanism shown in Fig. 1. The avidin solution was recycled

wice to immobilize until saturation. BABA and BBSA solu-ions were also prepared in 0.05 M Na2HPO4 buffer (pH 9)nd permeated at room temperature and 0.34 bar (5 psi) pres-ure with constant stirring at 300 rpm. Volumetric flow rate andumulative volume of permeates were measured at different timentervals. After immobilization of avidin and after permeating

300 S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310

Fig. 1. Immobilization of avidin (:NH2–R) in membrane containing acylanhydride group.

BABA, BBSA, BBSA + GG, the membrane matrix was washedwith 100 ml of 0.2 M NaCl solution to remove the reversiblyadsorbed molecules from the membrane. Although avidin isalso a protein, now onwards, unless mentioned otherwise, theterm “protein” will only be used to denote any biotinylated(BBSA, Tat)/non-biotinylated (BSA, GG) protein other thanavidin.

All spectrophotometric measurements were done by UV–visSpectrophotometer (Varian, Cary 300).The concentrations ofBABA were determined by measuring the absorbance directlyat 264 nm. The amount of avidin immobilized was deter-mined by measuring the concentration in the feed and perme-ate using Bradford protein assay technique [35]. Same proce-dure was followed for measuring pure BBSA concentrations.The salt-wash solutions were also analyzed to quantify thereversibly adsorbed species (avidin, BABA, BBSA). BBSA inmixture with GG was quantified by measuring the biotin con-tent of BBSA using HABA (2-(4′-hydroxyazobenzene)benzoicacid)–avidin complex method [25]. The linear range of detec-tion for BABA was 0.1–25 �g/ml and the analytical error wasless than 1% for 1–10 �g/ml and 1–15% for 0.1–1 �g/ml. Thelinear range of total protein analyses by Bradford protein assaywas 0.75–24 �g/ml and the analytical error was less than 1% for2–24 �g/ml and 1–10% for 0.75–2 �g/ml of protein solutionsin this research work. The lower limit of detection of biotinfor HABA–avidin complex method was 5 × 10−4 �moles/ml ofbc

s(pwtefwtaqtsf

bma

2.3. Separation and purification of tat from bacterial lysate(BL)

In order to obtain high quality Tat, it was genetically engi-neered to introduce biotin structure by gene fusion while cloningin the cell of E. coli vector. The detail of the production ofbiotinylated-Tat has been given elsewhere [4,36]. However, it isworth to mention here that after expressing Tat in E. coli vector,the bacterial cells were lysed using French pressure cell press(SLM Amimco), and then the bacterial lysate (BL) was cen-trifuged at 10,000 × g to remove cell debris and precipitates. Thebacterial lysate supernatant containing biotinylated-Tat alongwith other proteins was then treated separately by recognitionbased membrane separation and conventional packed-bead chro-matography to obtain pure Tat.

2.3.1. Recognition based membrane separationGenetically engineered Tat in BL contains a fusion pro-

tein and a biotin structure as shown in Fig. 2. This geneticallyengineered Tat was selectively separated from other proteinspresent in BL using the concept of avidin–biotin interaction inmembrane pores. The experimental steps for the pretreatment,separation, and purification of Tat from BL are presented inFig. 3.

2.3.1.1. Pretreatment of bacterial lysate (BL). Protein–proteinisct(tTs

taiear1I0ttt

iotin. To check the reproducibility, the analyses were tripli-ated.

Total protein present in membrane matrix can be clas-ified into two categories: (i) specifically attached proteinsavidin–biotin interaction), and (ii) non-specifically adsorbedroteins (protein–membrane and protein–protein interaction),hich consisted of reversibly and irreversibly adsorbed pro-

eins. The reversibly adsorbed proteins were quantified as statedarlier. In order to quantify the irreversibly adsorbed proteinsor the permeation of BBSA, separate permeation experimentsere carried out with non-biotinylated protein (BSA) under

he same conditions and concentrations. While calculating theccessibility of avidin sites, non-specifically adsorbed proteinuantity was deducted from the total proteins in membraneo estimate the specifically attached proteins. The amount ofpecifically attached Tat is directly obtained by cleaving withactor Xa.

To analyze the formation of the protein layers in the mem-ranes, pure water flow rate was measured through the bareembranes, after immobilization of avidin, and after perme-

tion of protein.

ntermolecular interactions cause aggregate formation. In BL,ome Tat gets entangled within these aggregates and they neverame in contact with the avidin as shown in Fig. 2. Forma-ion of these aggregates depends on the nature of the solutiondilution, ionic strength, etc.). The primary objective of bac-erial lysate pretreatment was to mitigate protein–protein andat–RNA interactions to increase the quantity of free Tat inolution.

In the previous work by our research group it was observedhat by diluting BL and applying a higher salt concentration theggregate formation between proteins can be reduced, therebyncreasing the available free Tat in the BL [4]. To verify theffect of dilution and salt concentration on the amount ofvailable free Tat in BL, two separate experiments were car-ied out. In one experiment, 1 ml of stock BL was diluted to×, 10×, 20×, 30× with 0.1 M (0.1 × 103 �moles/ml) NaCl.n another experiment, 1 ml of BL was diluted to 10× with, 0.1, 0.25, 0.5 M NaCl solutions. ELISA was carried outo measure the amount of Tat in solutions. The results ofhese experiments are discussed later; however, it is wortho mention here that the amount of available free Tat in BL

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 301

Fig. 2. Schematic of separation of Tat protein from bacterial lysate using avidin–biotin interaction in a modified membrane pore. Biotinylated-Tat (Bio-F-Tat) formsa complex (Tat-F-Bio-Av-Mem) with avidin-immobilized membrane (Av-Mem). Tat is then isolated by cleaving the Tat–Fusion protein bond with factor Xa. Someprotein adsorbs on the surface of the membrane and avidin due to non-specific interactions. Some Tat is entangled with other feed proteins.

was found to increase with increase in dilution and ionicstrength.

Further, it was observed that the basic region of Tat protein hasa high affinity to bind trans-activation responsive region RNAforming a Tat–RNA complex [37], which alters the binding affin-ity of Tat with the immobilized avidin due to steric hindrance.Hence, 25 �g RNAse/ml of bacterial lysate was added to degradeRNA and reduce the formation of the Tat–RNA complex.

2.3.1.2. Tat separation. Tat present in BL was separated bypermeating through an avidin-immobilized nylon membranesmatrix. Membrane matrix was consisted of four membranesheets arranged in stacked configuration to provide greater inter-nal surface area for avidin immobilization. Biotiylated Tat was

interacted with avidin and attached to the membrane matrix,while other proteins present in the mixture permeated through(Fig. 2).

In this research work, two experiments were carried out. Inone experiment, 10 ml and in another 15 ml of BL were dilutedto 300 ml with 0.5 M NaCl. Diluted BL solution was then per-meated at 0.34 bar pressure, and the permeate flow rate andcumulative volume were measured at different time interval. Toavoid oxidation of the proteins, DTT, and as protease inhibitorPMSF were added to all solutions used in the experiments.

2.3.1.3. Tat purification. Some proteins were adsorbed intothe system due to non-specific protein–avidin and protein–membrane interactions (Fig. 2). To remove them from the

acteri

Fig. 3. Experimental steps for the separation and purification of Tat from b al lysate (BL) using avidin–biotin interaction in stacked membrane matrix.

302 S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310

membrane matrix, it was washed with 150 ml of wash buffer(0.05 M Tris–HCl pH 8, 0.1 M NaCl, 1.6 × 10−5 M Triton X-100, and 0.031 M NaN3). The system was then preconditionedfor cleavage with 100 ml cleavage buffer (0.05 M Tris–HCl pH8, 0.10 M NaCl, 0.001 M CaCl2, and 0.031 M NaN3), followedby the cleavage of the purified Tat (along the fusion protein–Tatbond) from the membrane module by 20 �g factor Xa (cleav-age factor) per ml of cleavage buffer. SDS-PAGE and WesternBlot analysis were carried out to qualitatively determine andELISA was done to quantitatively determine the presence of Tatin bacterial lysate feed, permeate, retentate, wash buffer perme-ate, cleavage buffer permeate and the purified Tat in cleavagefactor (eluate).

For BL, to analyze the protein layer formed in the membranes,pure water flow rate through the 4-stack membrane matrix wasmeasured before and after the permeation of BL as done forother protein solutions. Then, to determine the contribution ofthe topmost membrane and the rest of the 3-stack membranesin the fouling, the topmost membrane was removed from thestack and water flow rate through the rest was measured underthe same conditions.

2.3.2. Packed-bead column chromatographySeparation of biotinylated-Tat was achieved by equilibrating

BL supernatant with 5 ml of avidin containing agarose beads(Ultralink Immobilized NeutrAvidin from Pierce Biotechnol-oobp[

3

aairtBc

3

3

igB

sbbm2w

buffer and allowed to react for 15 min at pH 7–9. Howeverin our research, for the single membranes, immobilization ofavidin was 3.8 × 10−2 to 5.1 × 10−2 �moles/ml bed volume.This corresponds to a biotin binding capacity (based onbiotin:avidin = 3.6:1) of 13.7 × 10−2 to 18.4 × 10−2 �moles/mlbed volume. Due to convective flow within the membrane pores,the avidin molecules could easily access the acylanhydridegroups, and therefore the amount of avidin immobilized wassignificantly higher for these experiments. The variation inthe amount of avidin immobilized was due to the presence ofdifferent amount of acylanhydride groups in different mem-branes and multiple attachment of single molecule of avidinwith their different amine groups. For first set of experimentwith 4-stack membranes (bed volume = 4 × 0.55 = 2.2 ml),the immobilization was 2.2 × 10−2 �moles avidin/mlbed volume, corresponding to biotin binding capacity of7.9 × 10−2 �moles/ml bed volume. In second set of exper-iment with 4-stack membranes, the immobilization was3.4 × 10−2 �moles avidin/ml bed volume (correspondingto biotin binding capacity of 12.2 × 10−2 �moles/ml bedvolume).

The effective thickness of the covalently immobilized avidinlayer in the membranes, δavidin, was calculated (applyingHagen–Poiseuille’s equation) using the water flow rate dataobtained before and after permeating avidin solution. Depend-ing on the amount of avidin immobilized, calculated δ

wcDwt2a0meptb

3

(stbrmm

ftbsofib

gy) in 30 ml of cell lysing buffer for 2 h. The binding capacityf the beads was 5 × 10−2 to 8 × 10−2 �moles biotin/ml ofeads. The purification and elution steps followed the samerinciple as in membrane separation and are given elsewhere4].

. Results and discussions

In this section, the results of immobilization of avidin andccessibility of immobilized avidin sites for different solutionsre described. Then, the results for the flux decline due to foul-ng of the membranes are presented, along with the results ofesistance offered by protein layer in the membranes. Finally,he efficiency of this technique to separate and purify Tat fromL is discussed and compared with that obtained for columnhromatographic separation.

.1. Accessibility of covalently immobilized avidin sites

.1.1. Immobilization of avidinFrom homogeneous phase avidin–biotin interaction exper-

ment it was determined that 1 mole of avidin, in homo-eneous phase, could bind 3.6 moles of biotin present inABA.

To study the variation in the amount of avidin immobilized,even sets of single membranes and two sets of 4-stack mem-ranes matrix were tested. Based on the information suppliedy the manufacturer, the acylanhydride groups present in aembrane (bed volume 0.55 ml) could covalently immobilize

.4 × 10−2 to 3.3 × 10−2 �moles of avidin/ml bed volume,hen spot loaded with 4000 �g/ml avidin solution in PBS

avidinas varied within 9–13 nm. The calculation of δavidin is dis-

ussed later in detail while explaining the results of fouling.ue to immobilization of avidin, 15–20% decrease in water fluxas observed for all the membranes. The initial flux through

he avidin-immobilized membranes (Jv0) was 14 × 10−4 to4 × 10−4 cm3/cm2 s (corresponds to a flow rate of 3–5 ml/minnd permeability of 4 × 10−3 to 7 × 10−3 cm3/cm2 s bar) at.34 bar. Based on the initial flux through avidin-immobilizedembrane and the porosity determined from water sorption

xperiment, the approximate initial linear velocity through theores was calculated to be 370–620 cm/h. At this linear velocityhe residence time through a 4-stack membranes system woulde 1.6–2.6 s.

.1.2. Permeation of biotin 4-amidobenzoic acid (BABA)In homogeneous phase, because of the ease of accessibility

Fig. 4(a)), 3.6 moles of biotin uptake was possible by the activeites of 1 mole of avidin. However, within the membrane pores,he biotin uptake by the immobilized avidin sites would be lessecause of the involvement of accessibility problems. In thisesearch, BABA was used as a probe to determine the maxi-um accessible sites of avidin, immobilized covalently withinembrane pores.The breakthrough curves obtained for the permeation of dif-

erent concentrations of BABA is shown in Fig. 5. The concen-ration is represented in terms of biotin. As expected, the initialreakthrough curve is steeper for higher concentration of BABAolutions due to higher amount of biotin permeated. It is alsobserved that Cp/Cf is never 0 at time > 0 (initially pores werelled with buffer) for any of the solutions. Due to the sufficientlyigger pores (∼0.42–0.43 �m) of avidin-immobilized MF mem-

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 303

Fig. 4. (a) Avidin in homogeneous phase. Approximately four sites are available for biotin. (b) Covalently immobilized avidin in membrane. Due to covalentattachment, avidin sites near membrane surface became blocked. (c) Multiple attachment of avidin molecules by different amine groups. (d) Blockage of avidin sitesby big molecules of protein (biotinylated as well as non-biotinylated).

Fig. 5. Comparison of breakthrough curves for the permeation of differentbiotin 4-amidobenzoic acid (BABA) solutions through avidin-immobilized(3.8 × 10−2 to 5.1 × 10−2 �moles/ml bed volume) single nylon membrane (porediameter = 0.45 �m) at 0.34 bar. Avidin functionalized membrane permeabil-ity = 4 × 10−3 to 7 × 10−3 cm3/cm2 s bar. Cp, concentration of biotin in perme-ate; Cf, concentration of biotin in feed.

branes and convective flow of permeate, significant amount ofbiotin has permeated through central region of the pores with-out even interacting with the avidin sites. This phenomenon isknown as “Core Leakage” (Fig. 2).

Even for this small biotinylated compound, BABA, theamount of biotin uptake (Table 1(a)) by the covalently immobi-lized avidin sites in the membrane was only half of the biotinuptake capacity of the same amount of avidin in homogeneousphase. This signifies that only half of the total active sites ofcovalently immobilized avidin in membrane were accessible byBABA. Thus, saturation biotin uptake by covalently immobi-lized avidin in membrane was considered to be that obtained byBABA, i.e. by considering biotin:avidin = 1.8:1. Based on that,for all biotinylated solutions used (Table 1, Figs. 7 and 8), nor-

malized accessibility of the covalently immobilized avidin siteswas defined as follows:

Normalized accessibility

= Moles of biotin uptake from biotinylated species

Saturation biotin uptake based on BABA

The normalized accessibility is very specific for a particulartype of membrane as the saturation biotin uptake by BABA canvary with functionalization techniques.

The possible reasons of lower accessibility of active avidinsites are following: (i) blockage of avidin sites near the walls ofthe membrane pores as shown in Fig. 4(b), (ii) multiple attach-ment of a single molecule of avidin by their different aminegroups as shown in Fig. 4(c), and (iii) conformation change onthe wall of membrane pores by protein–polymer interaction. Itwas also interesting to notice that the accessibilities were samefor all BABA solutions irrespective of the concentration of biotinin feed BABA (Table 1(a)). The non-dependency of accessibil-ity on concentration suggests that the small molecules of BABAdid not offer any steric hindrance to biotin moieties and did notblock the avidin sites.

3.1.3. Permeation of biotinylated-BSA (BBSA)

doaiBea

Table 1aAccessibility of avidin sites for the permeation of biotin 4-amidobenzoic acid (BAB0.34 bar

Avidin immobilized(×102 �moles)

Concentration ofBABA in feed(�g/ml)

Concentration ofbiotin in feed(×103 �moles/ml)

Bioavipha

2.4 9.6 25.0 8.62.1 3.2 8.3 7.62.3 0.6 1.56 8.3

Fig. 6 shows the breakthrough curves for the permeation ofifferent concentrations of BBSA solutions. The concentrationf biotin is obtained by considering each mole of BBSA is equiv-lent to 8 moles of biotin, as specified by the manufacturer. Thenitial breakthrough curve is steeper for higher concentration ofBSA due to the higher amount of biotin permeated, as observedarlier for BABA. “Core leakage” is also observed for BBSA,s for BABA.

A) through avidin immobilized nylon membrane (pore diameter = 0.45 �m) at

tin uptake capacity ofdin in homogeneousse (×102 �moles)

Biotin uptake byavidin (×102 �moles)

Normalizedaccessibility

4.3 13.8 14.2 1

304 S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310

Table 1bAccessibility of avidin sites for the permeation of biotinylated-BSA (BBSA), BBSA + gamma globulin (GG, non-biotinylated), and Tat in bacteria lysate (BL) throughavidin immobilized nylon membrane (pore diameter = 0.45 �m) at 0.34 bar

Avidin immobilized(×102 �moles)

Concentration ofbiotinylated species infeed (�g/ml)

Concentration ofbiotin in feed(×103 �moles/ml)

Biotin uptake byavidin (×102 �moles)

Saturation uptake ofbiotin based on BABA(×102 �moles)

Normalizedaccessibility

2.8BBSA

19.3 2.27 2.9 5.0 0.582.4 10.0 1.17 2.8 4.4 0.641.5 3.0 0.35 2.3 2.8 0.832.5 BBSA+GG (13 �g/ml) 10.0 1.17 2.5 4.5 0.554.8 Tat in BL (3864 �g/ml total protein) 14.30 1.72 3.0 8.6 0.357.6 Tat in BL (1120 �g/ml total protein) 14.50 1.74 2.3 13.6 0.17

Single membrane was used for BABA, BBSA and BBSA + GG, whereas, 4-stack membranes was used for permeation of Tat in BL.

Fig. 6. Comparison of breakthrough curves for the permeation of differentbiotinylated-BSA (BBSA) solutions through avidin-immobilized (3.8 × 10−2

to 5.1 × 10−2 �moles/ml bed volume) single nylon membrane (pore diam-eter = 0.45 �m) at 0.34 bar. Avidin functionalized membrane permeabil-ity = 5 × 10−3 to 7 × 10−3 cm3/cm2 s bar. Cp, concentration of biotin in per-meate; Cf, concentration of biotin in feed.

The normalized accessibility values given in Table 1(b) andplotted as a function of amount of biotin permeated in Fig. 7,show lower accessibilities for BBSA compare to BABA. It is nottrivial to explain the accessibilities of avidin sites for BBSA, asthe location and orientation of eight biotin moieties present permolecules of BBSA are unknown. It is possible, that the accessi-bilities for BBSA solutions were less because some of the biotin

Fig. 7. Comparison of accessibility of the avidin sites (3.8 × 10−2 to5.1 × 10−2 �moles/ml bed volume) immobilized in single nylon mem-brane (pore diameter = 0.45 �m) for different biotinylated-BSA (BBSA) solu-tions at 0.34 bar. Avidin functionalized membrane permeability = 5 × 10−3 to7 × 10−3 cm3/cm2 s bar.

moieties never exposed to avidin. It is also possible that insteadof all eight biotin moieties, BBSA molecules were attachedusing few biotin moieties only, making calculations of accessi-bility more difficult. In our calculations, a simple approach wastaken considering a BBSA molecule always attached with eightbiotin moieties. Fig. 7 depicts that as biotin moieties perme-ated through the avidin-immobilized membrane; they interactedwith avidin and bound to the membrane matrix. However, all thebiotin moieties could not access all the available avidin sites. Thebulky molecules of BBSA offered substantial steric hindranceto the biotin moieties present in it to access the avidin sites.Also, some BBSA molecules blocked the nearby avidin sites bynon-specific adsorption (Fig. 4(d)), thereby reducing the acces-sibility in comparison to BABA. It can also be observed fromFig. 7, that in contrast to BABA, accessibility shows a decreas-ing trend with increasing BBSA concentration throughout thepermeation experiment. This might be due to the fact that as theconcentration of BBSA in feed increases, the biotin moietiesexperienced increased steric hindrance from greater number ofprotein molecules present. The blockage of avidin sites by themolecules of proteins was also higher for concentrated BBSAsolution.

3.1.4. Permeation of BBSA + gamma globulinThe normalized accessibility of avidin sites for BBSA

pbitatp

tBotbtmBb

resent in a mixture with GG is shown in Table 1(b). Theiotin attachment, and hence the accessibility of avidin sitess less than that for pure BBSA with same biotin concentra-ion (1.17 × 10−3 �moles/ml biotin). In addition to the reasonslready discussed for BBSA, accessibility decreased further dueo the blockage of avidin sites by non-biotinylated protein (GG)resent in solution as well (Fig. 4(d)).

Fig. 8 compares the normalized accessibility as a function ofotal biotin permeated for different types of solutions (BABA,BSA, and BBSA + GG) containing comparable concentrationf biotin. The accessibility for BABA was always higher thanhe other two because of the unhindered access to avidin sitesy biotin moieties present in small BABA molecules. However,he accessibility was less for biotin moieties present in complex

olecules of proteins as discussed earlier. Between BBSA andBSA + GG, the initial biotin attachment was same, probablyecause the effect of non-biotinylated protein was insignificant

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 305

Fig. 8. Comparison of normalized accessibility vs. biotin permeatedfor biotin 4-amidobenzoic acid (BABA), biotinylated-BSA (BBSA) andBBSA + gamma globulin (GG, non-biotinylated protein) through avidin-immobilized (3.8 × 10−2 to 5.1 × 10−2 �moles/ml bed volume) single nylonmembrane (0.45 �m) at 0.34 bar. Avidin functionalized membrane permeabil-ity = 4 × 10−3 to 5 × 10−3 cm3/cm2 s bar.

initially. But, finally accessibility decreased for BBSA + GG dueto the increased steric hindrance and blockage of avidin sites bythe non-biotinylated protein.

3.1.5. Permeation of bacterial lysate (BL)From Table 1(b) it can be observed that the avidin site acces-

sibility is least for the permeation of BL. Although concentrationof biotin (biotin:monomeric Tat = 1:1) present in BL was compa-rable with 0.6 �g/ml of BABA, 10 �g/ml of BBSA and 10 �g/mlof BBSA + 13 �g/ml of GG; 98–99 wt.% of non-biotinylatedprotein present was responsible for the decreasing accessibility.The difference in accessibility for the two cases of BL, particu-larly the lower accessibility for more dilute solution, is difficultto explain. However, due to the complex nature of the mixture itcan be assumed that some other factor, like interactions betweenproteins present in BL, might have overshadowed the advantageof lower concentration of proteins in solution.

Fig. 9. Comparison of normalized flux (Jv/Jv0) for permeation of BSA, BBSA,BBSA + gamma globulin (GG) and bacterial lysate (BL) through avidin-immobilized nylon membranes (pore diameter = 0.45 �m) at 0.34 bar. Normal-ized flux = ratio of flux at any time of permeation to initial flux. Jv0 = 14 × 10−4

to 24 × 10−4 cm3/cm2 s.

3.2. Study of associated fouling

Fouling in the membranes due to permeation of proteins canbe observed by the flux decline curves shown in Fig. 9. Due tothe presence of higher amount of protein (attached + adsorbed) inthe membrane (Tables 2a and 2b), reduction of flux increases asthe concentration of protein in feed increases (except 10 �g/mlBBSA without avidin and 10 �g/ml BSA). For BL (3864 �g/mltotal protein) the flux decline (85%) is highest because of max-imum amount of protein in membrane matrix.

Membrane pores have experienced fouling because of theadsorption of proteins due to different interactions (hydrophobic,electrostatic, etc.). Some protein molecules formed aggregatescaused by protein–protein interaction (for example, Tat and BSAare rich in cysteine residues, so prone to form aggregates by S–Slinkages) and deposited in the pores of the membrane. In theexperimental pH, the immobilized avidin molecules contained

Table 2aCalculated resistance and thickness of protein layer formed due to the permeation of BSA, biotinylated-BSA (BBSA), BBSA + gamma globulin (GG) through avidinimmobilized single nylon membrane (pore diameter = 0.45 �m) at 0.34 bar

Concentration of protein in feed Total amount of proteinin membrane (�g)

Rp (×10−10 cm−1) δp (nm)

10 �g/ml BSA 256 1.25 6.03 �g/ml BBSA 307 1.53 12.0

1 2.37 15.511

R

TC tion o(

Cp

×10−

31

R embrp

0 �g/ml BBSA 4939.3 �g/ml BBSA 6530 �g/ml BBSA + 13 �g/ml GG 678

p, resistance of protein layer; δp, effective thickness of protein layer.

able 2balculated resistance and thickness of protein layer formed due to the permea

pore diameter = 0.45 �m) at 0.34 bar

oncentration of totalrotein in BL (�g/ml)

Total amount of proteinin membrane (�g)

Rp = R1p + R3p (

864 10251 12.32120 7712 8.32

p, protein layer resistance in 4-stack membranes; R1p, resistance in topmost mrotein layer.

2.63 20.02.75 27.0

f bacterial lysate (BL) through avidin immobilized 4-stack nylon membranes

10 cm−1) R1p (×10−10 cm−1) R3p (×10−10 cm−1) δp (nm)

8.58 3.74 68.05.61 2.70 61.0

ane; R3p, resistance in rest of the 3-stack membranes; δp, effective thickness of

306 S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310

some net positive charge, whereas, BBSA molecules were neg-atively charged. So, some protein molecules were attached byelectrostatic interaction also. Reversibly adsorbed moleculeswere removed by salt washing. However, significant amount ofproteins were adsorbed irreversibly, particularly for the higherconcentration of protein solution, and hence, the recovery of per-meate flux was not to the extent of the initial flux through avidin-immobilized membranes. Based on the initial flux throughavidin-immobilized membrane, the flux recovery was 90% for10 �g/ml BSA and 70–80% for 19.3–3 �g/ml BBSA. The fluxrecovery (25%) to significant extent was not possible for BL bywashing with salt solutions (wash buffer and cleavage buffer),which proves highly irreversible nature of fouling for BL.

To study the contribution of membrane itself on flux decline inabsence of avidin–biotin interaction, control experiments wereperformed by permeating BBSA through membranes withoutavidin. From Fig. 9 it is observed that flux decline (17%) for10 �g/ml BBSA without avidin is less than flux decline (55%)for 10 �g/ml BBSA with immobilized avidin. Lower amount ofprotein adsorption (120 �g compared to 493 �g) in membranedue to the absence of avidin–biotin and/or avidin–protein inter-action was the reason behind this observation. This suggests thatmembrane itself has a minor role, whereas, avidin–biotin and/oravidin–protein interactions play a critical role in fouling.

The results for two different BSA experiments (10 and3864 �g/ml) are also presented in Fig. 9 for comparison withBlpopFt(spd

taglfcpd

t(r

J

a

Q

where r is the radius of the bare membrane pore; µ the viscosityof solution; L the thickness of the membrane; �P the trans-membrane pressure drop; τ the tortuousity of the membrane;porosity, ε = NPπr2τ/Am = 0.32; NP the total number of poresin the membrane; Am, external membrane surface area and theintrinsic membrane resistance, Rm = 8τL/εr2.

For permeation of proteins, the volumetric flux, Jv, throughthe fouled membrane can be described using Darcy’s Law asfollows [39]:

Jv = �P

µ(Rm + Rp)(3)

where, Rp is the resistance offered by the layer of proteins presentin the membrane.

Rearranging Eqs. (1)–(3) the expressions for Rm and Rp canbe obtained as follows:

Rm = �P

µJw(4)

Rm + Rp = �P

µJv(5)

Assuming Np remains same and the proteins form an effectiveuniform thickness of layer throughout the pores of the mem-brane, Eq. (2) could be reproduced for the volumetric flow rateof pure water through the fouled membrane, Qw1, by substitutingtoa

r

Ebw

ttc

δ

δ

Eta(tptgtnfmlaδ

BSA and BL. Fouling is less for 10 �g/ml BSA (0% biotiny-ated protein) than that for 10 �g/ml BBSA (100% biotinylatedrotein), suggesting that in presence of significant concentrationf biotinylated protein, fouling is not dependent entirely on totalrotein in feed but depends strongly on the biotin molecules.or BBSA, due to the avidin–biotin interaction the amount of

otal protein present in membrane is higher than that for BSATable 2a), which has caused the higher flux drop. It is also pos-ible that, due to complex orientation of eight biotin moleculesresent per molecules of BBSA, it offered higher steric hin-rance to the flow than non-biotinylated-BSA.

On the other hand, similar flux decline trend is observed forhe permeation of 3864 �g/ml BSA (0% biotinylated protein)nd BL (3864 �g/ml total protein, 1% biotinylated protein), sug-esting that in absence of significant concentration of biotiny-ated protein fouling is dependent entirely on total protein ineed. This observation has also pointed out that fouling, in thisase, was not dependent on the type of non-biotinylated proteinresent, as BSA and mixture of proteins (BL) have same fluxecline trend.

Assuming all pores in the membrane have the same radius,he volumetric permeate flux (Jw) and the volumetric flow rateQw) of pure water through a micro porous membrane can beepresented by Hagen–Poiseuille’s equation as [38]

w = εr2�P

8µτL= �P

µRm(1)

nd,

w = NPπr4�P

8µL(2)

he radius of the bare membrane pore (r) with the effective radiusf the fouled membrane pore (rP). Now, rP can be representeds

P = r

(Qw1

Qw

)0.25

(6)

q. (6) can also be used to calculate the effective radius of mem-rane pores after immobilization of avidin (ravidin), using theater flow rate data obtained after the immobilization.The effective thickness of immobilized avidin layer (δavidin),

he effective thickness of protein layer (δp) and the effectivehickness of total protein (including avidin) layer (δT) can bealculated as

T = r − rP, δavidin = r − ravidin, and

P = δT − δavidin = ravaidin − rP (7)

qs. (4) and (5) were used to calculate the equivalent resis-ance (Rp) offered by the effective protein layer and Eqs. (6)nd (7) were used to calculate the thickness of the protein layerδp) present in the membrane pores after the permeation of pro-ein and salt washing. Due to the high concentration of proteinsresent in BL, surface fouling occurred to some extent along withhe adsorption of proteins within the pores. However, in order toet a rough estimate of the effect of protein layer on the fouling,he same assumption of “formation of an effective uniform thick-ess of protein layer throughout the pores of the membrane” wasollowed for BL also. For BL, it was also assumed that 4-stackembranes system obeys resistance-in-series model. The calcu-

ated values of Rp and δp for different concentrations of proteinsre presented in Tables 2a and 2b. It can be observed that Rp andp increase with increase in protein concentration in feed, due to

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 307

Fig. 10. Comparison of effective thickness of protein layer within the mem-brane while permeating BSA, BBSA, BBSA + gamma globulin (GG) and bac-terial lysate (BL) through avidin-immobilized nylon membranes (pore diame-ter = 0.45 �m) at 0.34 bar. Initially the membranes had the immobilized avidin,above which the protein started adsorbing to form the layer.

the presence of higher amount of protein in the membrane. TheRp and δp for both cases of BL (Table 2b) are much higher thanthat for BSA, BBSA, and BBSA + GG (Table 2a). For BL, to cal-culate the separate contribution of the topmost membrane (R1p)and the rest of the 3-stack membranes (R3p) in final protein layerresistance (Rp), the data collected by removing topmost mem-brane along with that for 4-stack membrane system were used.It can be observed from Table 2b that approximately 70% of theRp was contributed by R1p for both the BL experiments.

The increase in the effective thickness of protein layer duringthe permeation of BSA, BBSA, BBSA + GG, and BL were cal-culated using same type of correlation as Eqs. (6) and (7). Thecalculated values of effective thickness are plotted in Fig. 10as a function of V. Starting from zero thickness, the layer builtup sharply as protein molecules were attached by avidin–biotininteraction, as well as adsorbed by non-specific protein–avidininteraction on the membrane pores. Later, the formation of layerwas gradual due to the slower rate of attachment and adsorption,and finally it leveled off.

The surface morphology of the membranes, obtained by SEMimages (Fig. 11), shows the effect of surface fouling on fluxdecline. Fig. 11(a) and (b) show the surface morphology ofthe bare membrane and avidin-immobilized membrane, respec-

tively. Surface fouling is not observed for avidin-immobilizedmembrane. The membrane for BBSA experiment, as shown inFig. 11(c), experienced insignificant surface fouling because oflower concentration of protein in feed side. Substantial foulingis observed (Fig. 11(d)) only in some places of the surface ofthe topmost membrane used for BL permeation. So, it can beinferred that the fouling is merely on the surface of membraneas shown by SEM images. However, because of high proteinconcentration for BL (total protein concentration = 3864 �g/ml)the aggregated proteins were deposited on some places of mem-brane surface causing some surface fouling.

3.3. Separation and purification of Tat

Fig. 12(a) and (b) show the effect of dilution and salt con-centration of BL on available free Tat as analyzed by ELISA.Normally with dilution the total amount of a component presentin a solution remains same (the concentration decreases). In caseof Tat in BL, it is observed that the total amount of available freeTat in diluted BL increased with increase in dilution as shown inFig. 12(a). This observation suggests that at higher concentrationof total protein, Tat was entangled within the aggregates formed.As dilution increased, due to decrease in protein–protein inter-action Tat was released from the aggregates. Similarly, due tothe shielding effect of ions, the interaction between the proteinsdecreased at higher salt concentration and more amount of Tatw

mlTwwedamaS[

F memm A andn = 386

ig. 11. SEM images (6 k magnification) of the surface of the nylon based MFembrane; (c) single nylon membrane through which 10 �g/ml biotinylated-BS

ylon membranes matrix through which bacterial lysate (protein concentration

as released in BL as shown in Fig. 12(b).SDS-PAGE fractionates proteins in a mixture based on their

olecular weight and shows specific band for different molecu-ar weight proteins present. Fig. 13(a) and (b) show that for bothat separation experiments, feed, permeate and retentate streamsere consisted of different types of proteins along with Tat. Theash buffer and cleavage buffer did not contain Tat protein. The

luate stream, which is basically the purified Tat stream, have aistinct band near 13–14 kDa molecular weight range, which isproof of the isolation of pure Tat in our research. Although theolecular weight of Tat monomer is 8335 Da, the band obtained

t 13–14 kDa might be due to the strong interaction betweenDS and the highly basic polypeptide sequence present in Tat4]. For comparison, the SDS-PAGE for the purified Tat stream

branes (pore diameter = 0.45 �m). (a) Bare membrane; (b) avidin-immobilizedsalt solution were permeated at 0.34 bar; (d) topmost membrane of the 4-stack

4 �g/ml), wash buffer and cleavage buffer were permeated at 0.34 bar.

308 S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310

Fig. 12. Effect of dilution and ionic strength on free available Tat present in bacterial lysate. (a) Effect of dilution on free available Tat by diluting bacterial lysatewith 0.1 M (0.1 × 103 �moles/ml) NaCl. (b) Effect of ionic strength on free available Tat by diluting bacterial lysate (DF = 10×) with different concentrations ofNaCl.

Fig. 13. SDS-PAGE images of different streams for the separation of Tat from bacterial lysate using avidin–biotin interaction in avidin-immobilized 4-stack nylonmembranes (F, feed; P, permeate; R, retentate; WB, wash buffer; CB, cleavage buffer; E, Tat eluate). (a) Concentration of total protein in feed = 3864 �g/ml, avidinimmobilized = 2.2 × 10−2 �moles/ml bed volume. (b) Concentration of total protein in feed = 1120 �g/ml, avidin immobilized = 3.4 × 10−2 �moles/ml bed volume.Extreme two right hand columns show the SDS-PAGE for Tat eluate obtained by column chromatographic separation (CC, column chromatography).

(eluate) obtained by conventional packed-bead column chro-matographic separation is displayed in the extreme two rightcolumns of Fig. 13(b). For analyzing membrane and columnchromatographically purified Tat by SDS-PAGE, the eluateswere loaded in equal volumes. It can be observed that the Tateluate obtained by conventional column chromatographic sepa-

ration shows several other bands due to the presence of proteinimpurities.

Western Blot analysis has also been used to compare thepurity of Tat protein obtained by packed-bead column chro-matographic and membrane separation. Since, response to aspecific antibody is utilized to analyze a sample in Western Blot;

Fig. 14. Western Blot analysis of different streams for the separation of Tat from bacterial lysate using avidin–biotin interaction in avidin-immobilized 4-stack nylonmembranes (F, feed; P, permeate; R, retentate; WB, wash buffer; CB, cleavage buffer; E, Tat eluate). (a) Concentration of total protein in feed = 3864 �g/ml, avidinimmobilized = 2.2 × 10−2 �moles/ml bed volume. (b) Concentration of total protein in feed = 1120 �g/ml, avidin immobilized = 3.4 × 10−2 �moles/ml bed volume.Extreme two right hand columns show the Western Blot for Tat eluate obtained by column chromatographic separation (CC, column chromatography).

S. Datta et al. / Journal of Membrane Science 280 (2006) 298–310 309

it is more sensitive to the purity of the product. As seen fromFig. 14, the purified Tat obtained from membrane separationcontains a distinct band near 13 kDa, indicating the presenceof pure monomeric form of Tat, whereas the Tat from packed-bead column chromatographic separation contains several otherbands ranging from 10 to 80 kDa, indicating the presence ofpolymeric forms of Tat, which are unwanted for the therapeuticusages of the Tat protein.

The amount of Tat recovered by recognition based membraneseparation was determined by ELISA of the eluate stream. Totalamount of purified Tat isolated were 246 and 190 �g, respec-tively, for two Tat separation experiments (eluate concentration15 �g/ml for both cases), which exhibits 5–6% recovery of Tatfrom BL. The % recovery implies that substantial amount of Tat(95%) is still present in other streams along with the impurities.

4. Conclusions

Recognition based separation technique was applied to obtainhigh quality Tat protein from bacterial lysate supernatant usingavidin–biotin interaction in modified stacked MF membranes.Tat protein obtained by this technique was superior in qualitythan that obtained by conventional packed-bead column chro-matographic separation as observed by SDS-PAGE and West-ern Blot analysis. The efficiency of separation depends on theaccessibility of the covalently immobilized avidin sites by thebmtfiitmaboTpatsatdpvttTbrvobmas

able proteins from mixtures using avidin–biotin interaction inmodified membranes.

Acknowledgements

We acknowledge NIH for financial support of this research(supported by NIH grants P20RR15592 and P01MH070056).We also acknowledge Dr. Aaron Hollman (Alumni of Universityof Kentucky) for his experimental contributions.

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