Inexpensive and Generic Affinity Purification of Recombinant Proteins Using a Family 2a CBM Fusion...

Inexpensive and Generic Affinity Purification of RecombinantProteins Using a Family 2a CBM Fusion Tag

Beatriz Rodriguez,† Mojgan Kavoosi,† Ju1 rgen Koska,† A. Louise Creagh,†Douglas G. Kilburn,‡ and Charles A. Haynes*,†

The Biotechnology Laboratory and the Protein Engineering Network of Centres of Excellence, the Department ofChemical and Biological Engineering and the Department of Microbiology and Immunology, The University ofBritish Columbia, Vancouver, British Columbia, Canada V6T 1Z3

The selective binding of the family 2a carbohydrate binding module (CBM2a) ofxylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosicsubstrates is shown to provide a new, cost-effective affinity chromatography systemfor purification of recombinant protein. Genetic linkage of CBM2a to a target protein,in this case protein A from Staphylococcus aureus, results in a fusion protein thatbinds strongly to the particulate-cellullose resin Avicel PH101 and retains the biologicalactivity of the fusion partner. Affinity purification of protein A-CBM2a from thesupernatant of a recombinant E. coli JM101 culture results in a product purity ofgreater than 95% and a product concentration factor of 34 ( 3. Measured columnparameters are combined with one-dimensional equations governing continuity andintraparticle diffusion to predict product breakthrough curves with good accuracy overthe range of realistic operating conditions. Peak spreading within the column iscontrolled by intraparticle diffusion for CBM2a and by a combination of film masstransfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein.

IntroductionThe purification of recombinant protein products from

fermentation broths remains one of the most difficult andexpensive tasks in biotechnology (1). Affinity separationsare attractive because they can potentially replace low-yield multistep purification procedures with a singlehighly selective adsorption step that simultaneouslyconcentrates and purifies the product (2). Advances inrecombinant DNA technology have allowed the power ofhigh-affinity biospecific interactions to be genericallyapplied to the concentration and purification of recom-binant proteins. Recombinant hybrids containing an N-or C-terminally fused affinity polypeptide tag that selec-tively binds to a complementary ligand immobilized ontoa suitable chromatographic matrix are now widely usedto facilitate the purification of target proteins or peptides.In addition to allowing the rapid purification of a targetprotein, affinity tags can also improve product solubility,increase in vivo proteolytic stability, and control productlocalization in or secretion from the expression host (3).Skillful design of the fusion-tag/immobilized-ligand paircan therefore provide a robust and generic method forefficient production and high-resolution affinity purifica-tion of recombinant protein targets.

Numerous affinity tag systems have been developed(2), including glutathione S-transferase from Schistosomajaponicum (4), the S-peptide from ribonuclease A (5),Staphylococcus aureus protein A and its synthetic two-domain variant (6, 7), and various polyamino acid affinitytags such as polyhistidine that form the basis of im-

mobilized metal-ion affinity chromatography (IMAC) (8).Although each of these affinity tag systems has beenshown to offer particular advantages, none has foundwidespread use in production-scale applications, due inpart to the high cost of the associated affinity matrix thatarises from the complex chemical modifications requiredto cross-link the solid support or to graft the affinity-tagreceptor to the resin surface. The relatively low toleranceof many of these affinity resins to repeated processingand sanitization (CIP) cycles also limits their use at theproduction scale.

Cellulose has a number of properties that make itdesirable as a generic affinity chromatography matrix.It is inexpensive, has good mechanical and chemicalstability, and has low nonspecific affinity for mostproteins. A range of cellulose and modified-celluloseresins in particulate and monodisperse spherical formsare commercially available for size-exclusion chromatog-raphy (SEC) applications. Moreover, Ladisch and co-workers have developed a novel set of spiral-wound SECresins using tightly rolled cotton fabrics (9), while thewide availability of inexpensive cellulose-based mem-branes and filters suggests that cellulose may find usein membrane-based separations, including membranechromatography. Finally, cellulosics are safe and havebeen approved for many pharmaceutical and human uses(10, 11).

This paper describes a generic and inexpensive affinitypurification technology based on high-level recombinantexpression in E. coli of fusion proteins containing acarbohydrate binding module (CBM) attached to eitherthe N- or C-terminus of the target protein or polypeptide.CBMs are discrete protein modules found in a largenumber of carbohydrolases and a few non-hydrolyticproteins. The more than 200 CBMs found in â-1,4-

* To whom correspondence should be addressed. Email: [email protected].

† Department of Chemical and Biological Engineering.‡ Department of Microbiology and Immunology.

1479Biotechnol. Prog. 2004, 20, 1479−1489

10.1021/bp0341904 CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical EngineersPublished on Web 07/01/2004

glucanases from a wide range of bacterial and fungalorganisms can be classified into 27 unique families, 17of which are known to contain members that bindcellulose (12). CBMs from different families are knownto have different binding affinities and binding specifici-ties (13). For example, CfXyn10A-CBM2a (hereafterreferred to as CBM2a), a family 2 CBM from xylanase10A of Cellulomonas fimi, binds crystalline regions ofinsoluble cellulose with a dissociation constant in the lowmicromolar range (14, 15). In contrast, the family 4 CBM,CfCel9B-CBM4-1 from endoglucanase 9B of C. fimi, bindsamorphous cellulose and water-soluble cello-oligosaccha-rides but shows no affinity for crystalline cellulose (16).In this work, CBM2a is used as a capture tag forrecombinant expression in E. coli and affinity purificationof S. aureus protein A on a column packed with particu-late semicrystalline cellulose particles. A one-dimensionalmodel is applied to the column to establish a simple andeffective method for describing flow, transport, andbinding properties in the column and for predictingproduct breakthrough.

Materials and MethodsMaterials. CF1 particulate microcrystalline cellulose

and Sigmacell Type 50 SEC resin were purchased fromSigma Chemical Co. (Mississauga, ON, Canada). AvicelPH101 was purchased from FMC International (LittleCounty Island, Cork, Ireland). Avicel is a particulatesemicrystalline cellulose preparation obtained from woodfibers by partial acid hydrolysis followed by spray dryingof the washed slurry (17). Prior to use, Avicel PH101powder was sieved to obtain a set of particle size fractionsbased on the average diameter dp of the particle: fraction1 (350 mesh size), dp < 45 µm, fraction 2 (250 mesh size),45 µm < dp < 75 µm, fraction 3 (200 mesh size), dp > 75µm. The range of particle diameters and the average dpof each fraction was determined by imaging 100 particlesof the sieved Avicel using an inverted microscope (Axio-vert 100, Carl Zeiss, Oberkochen, Germany) equippedwith a monochrome solid-state camera (COHU Inc., SanDiego, CA) connected to a real-time frame-grabber (Vi-sionplus-AT, Imaging Technology Inc., Woburn, MA).Particle volumes were then calculated assuming a prolateellipsoid geometry from which the mean particle diameterof the sphere was obtained. The mean diameter dp offraction 2, the fraction chosen for our column chroma-tography system, was 58 ( 12 µm.

CBM2a Production and Purification. A portion ofthe CBM2a used in these studies was obtained as a giftfrom Repligen Sandoz Chemicals (Lexington, MA) as a2 g L-1 aqueous solution and was maintained in thatstate at 4 °C until use. The remainder was produced byfermentation. The gene fragment encoding CBM2a wassubcloned into the pTugK07 vector and expressed inEscherichia coli strain JM101 according to the protocolsof Hassenwinkle et al. (18). The pTugK vector containsthe leader sequence for xylanase 10A of C. fimi, whichdirects the recombinant protein to the periplasm of E.coli. Hassenwinkle et al. (18) demonstrated that signifi-cant leakage of CBM2a from the periplasm into theculture supernatant occurs at high levels of expression.CBM2a used in this work was therefore recovered fromthe culture supernatant by centrifuging the final cultureat 4 °C and 10,000 rpm for 20 min and discarding thecell paste. The clarified supernatant was then mixed withan equal volume of sieved Avicel PH101 and allowed toequilibrate at 4 °C for 4 h.

The CBM2a-loaded Avicel was recovered by centrifu-gation at 15,000 rpm for 15 min. The Avicel pellet was

resuspended in 50 mM phosphate buffer, recovered, andthen washed with four volumes of the buffer. TheCBM2a-loaded Avicel in 60 mL of buffer was then pouredand packed into a Pharmacia XK-20 column, washed withtwo column volumes of phosphate-buffered saline, andfinally washed with one column volume of 50 mMphosphate buffer. Pure CBM2a was then eluted from thecolumn with either pure water or 6 M guanidiniumhydrochloride. CBM2a eluted using GuHCl was rena-tured by diafiltering the 6 M solution against five liquidvolumes (40 min) of 50 mM phosphate buffer (pH 7)containing 0.01% sodium azide. A 350 mL Amiconultrafiltration cell containing a 1 kDa molecular-weight-cutoff membrane was used to exchange the solvent. Therecovered CBM2a (94 ( 5% yield) was stored in thedialysis buffer at 4 °C until use.

Production of Protein A-CBM2a Fusion. The genefragment encoding CBM2a and the proline-threonine-richlinker sequence that precedes it in xylanase 10A of C.fimi (19) were introduced into the plasmid pRIT5 (Am-ersham-Pharmacia Inc., Sweden), which contains theleader sequence and IgG binding domains of S. aureusprotein A. The resulting pSACBM2a plasmid encodingthe protein A-CBM2a fusion (molecular weight 42 kDa)was cloned into E. coli JM101. Stock cultures of therecombinant host were prepared as described by Has-senwinkle et al. (18) in the presence of 0.05 mg mL-1

kanamycin and stored at -70 °C.Single colonies were used to seed a 10-mL inoculum

culture grown at pH 7 and 37 °C in an optimized M9minimal media containing 11.76 g L-1 Na2HPO4, 5.88 gL-1 KH2PO4, 0.5 g L-1 NaCl, 1.4 g L-1 NH4Cl, and 0.24g L-1 MgSO4 and supplemented with 0.2% glucose. Atrace metal solution (0.5 mL L-1) was also added andcontained the following salts per liter of water: 40 g ofCaCl2‚H2O, 10 g of MnSO4‚H2O, 10 g of AlCl3‚6H2O, 4 gof CoCl2‚6H2O, 2 g of ZnSO4‚7H2O, 2 g of Na2MoO4‚2H2O,1 g of CuCl2‚2H2O, 0.5 g of H3BO3, and 0.05 g of FeSO4‚7H2O. A 100 µL solution containing 0.5 g L-1 L-proline,0.34 g L-1 thiamine, and 100 µg mL-1 kanamycin wasadded to the culture through a 0.22 µm sterile filter.

The inoculum was grown to an OD (600 nm) of 1 andthen added to 400 mL of fresh media in a 2-L shake flaskto culture at 37 °C for up to 37 h. Glucose was periodicallyadded to the culture to a final concentration of 2 g L-1,and proline (0.25 g L-1) and kanamycin (50 µg mL-1) wereadded at 23 h culture time. Protein A-CBM2a is consti-tutively expressed in this construct, and a large fractionof the fusion-protein product is exported by leakage intothe culture supernatant. The final culture supernatantwas recovered by centrifugation at 10,000 rpm for 10 minat 4 °C. The concentration of protein A-CBM2a in theculture supernatant was measured by densitometry(Molecular Dynamics; Palo Alto, CA) scans of SDS PAGEgels using purified protein A-CBM2a as a standard.

Glucose concentrations in culture supernatants weremeasured using a Beckman Glucose Analyzer II cali-brated against a 1.50 g L-1 standard.

Binding Isotherms and Thermodynamics Mea-sured by Isothermal Titration Calorimetry. Equi-librium binding isotherms for CBM2a and for proteinA-CBM2a adsorption to sieved Avicel (fraction 2) weredetermined at pH 7 and 20 °C by the depletion methodaccording to the protocol described by Creagh et al. (14).The concentration of unbound protein remaining in thesupernatant was determined by absorbance at 280 nm.The extinction coefficients for CBM2a and proteinA-CBM2a are 2.31 and 0.8 mL cm-1 mg-1, respectively.All isotherm measurements were carried out in duplicate,

1480 Biotechnol. Prog., 2004, Vol. 20, No. 5

and errors in regressed equilibrium binding parametersrepresent mean standard deviations.

Affinity Chromatography. All chromatograms weremeasured on a Pharmacia Inc. (Uppsala, Sweden) FPLCsystem equipped with two P-500 reciprocating pumps,an 8-port mixing and injection valve, a UV-MII flowspectrophotometer (280 nm), and a FRAC-200 fractioncollector. The affinity column was prepared by mixingapproximately 3 g of sieved Avicel (fraction 2) with 50mM phosphate buffer (pH 7) to a volume of 7.5 mL. Thewell-mixed slurry was poured into an inclined (60° fromhorizontal) 1-cm ID XK-10 column (Pharmacia, Inc.) witha total working volume of 8 cm3. The column was thenrepositioned vertically and, following settling of the resinto form a clear interface, the outlet flow adaptor wasinserted and compressed onto the cellulose to fingertightness. The column was then equilibrated with de-gassed loading buffer (50 mM phosphate buffer, 0.05%NaN3, pH 7) at a flowrate between 0.2 and 1.0 mL min-1

until a stable baseline was observed.Breakthrough curves were measured by first equili-

brating the column with degassed loading buffer at afixed specified flow rate. The sample, either purifiedCBM2a or culture supernatant containing CBM2a(MW ) 11 kDa) or protein A-CBM2a (MW ) 42 kDa),was then loaded isocratically onto the column untilcomplete sample breakthrough was observed. Up to 1 Lof culture supernatant containing either 242 ( 48 µMCBM2a or 51.7 ( 17.3 µM protein A-CBM2a was loadedonto the 7.5-mL packed Avicel column at a superficialvelocity u of 2.12 × 10-4 m s-1 to obtain completebreakthrough.

All elution chromatograms for protein A-CBM2apurification from clarified culture supernatant werecollected in 1-mL fractions for analysis. Following loadingto 1% breakthrough, the column was washed for 1 h withphosphate-buffered saline (pH 7) and then washed foran additional hour with 50 mM phosphate buffer (pH 7).Bound protein (protein A-CBM2a) was eluted withdistilled water.

CBM2a concentrations in elution chromatograms weredetermined by UV absorbance at 280 nm using anextinction coefficient of 2.31 ( 0.04 mL cm-1 mg-1

determined by standard calibration. Concentrations ofprotein A-CBM2a in culture supernatants and in columneluent were determined by an antibody-based dot-blotassay. A 2-µL sample was spotted on Whatman 1 thin-film chromatography (TLC) paper preblocked for 1 h with3% bovine serum albumin (BSA) in phosphate-bufferedsaline (pH 7) containing 0.05% Tween-20. The proteinA-CBM2a-loaded TLC paper was then incubated for 1h at room temperature with rabbit anti-â-CBM2a anti-bodies (1:500 in PBS containing 0.05% Tween-20 and0.5% BSA) raised against CBM2a of C. fimi xylanase 10A.The membrane was then washed three times with PBS(0.05% Tween) and incubated for 1 h at room temperaturewith goat anti-rabbit IgG coupled to horseradish peroxi-dase (1:7000 in pBS containing 0.05% Tween and 0.5%BSA). The concentration of protein A-CBM2a was thendetermined using the enhanced chemiluminescence kitfrom Amersham Biotech.

Model Development

Continuity and Solute Uptake Equations. Giventhe radial and angular symmetry of our affinity column,we have adopted a standard one-dimensional continuityequation to describe changes in target protein concentra-

tion c with time t within a differential volume elementalong the column axis z

where DL is the axial dispersion coefficient (m2 s-1), u isthe interstitial liquid velocity (m s-1), ε is the interstitialvoid fraction, and s is the target protein concentration(mol m-3) within the stationary phase given by

where â is the porosity of the stationary phase, ci is thesolute concentration (mol m-3) in the pore liquid of thestationary phase, Fp is the density of the stationary phase(kg m-3), and qi is the specific concentration of proteinadsorbed on the stationary phase matrix (mol kg-1).

The flux No (mol m-2 s-1) of protein into the sphericalcellulose particles of radius R (m) is given by

where Dp (m2 s-1) is the effective diffusivity of the solutewithin the pore liquid of the stationary phase (based onthe entire particle volume), kf is the mass-transfercoefficient (m s-1) for protein transport within the fluidfilm, and â is the effective porosity of the stationary phase(i.e., the intraparticle pore fraction accessible to thesolute).

Solute transport within the spherical porous AvicelPH101 particles is given by

where r is the radial position (m) within the particle.Convection is not considered within the particle volume,and we have assumed that intraparticle solute concen-trations are angularly independent at all radial positions.

Binding Isotherm. Fluorescence recovery after pho-tobleaching studies by Jervis et al. (20) show that boundCBM2a can diffuse in two dimesions on cellulose. How-ever, depending on the surface concentration of CBM2aand the method of cellulose preparation, between 30%and 40% of the bound CBM2a is immobile on the cellulosesurface. Therefore, there exists the potential for stericexclusion of binding lattice sites on the sorbent surfacesuch that not all potential binding sites are filled atplateau adsorption levels (i.e., the surface jamming limitis less than the total number of solvent-exposed bindingsites). This results in a nonlinear Scatchard plot for theadsorption of CBM2a on the Avicel affinity chromatog-raphy resin at 20 °C (pH 7; 50 mM phosphate buffer).

Jin et al. (21) have derived a binding kinetics modelthat incorporates surface exclusion effects. Bound proteinconcentrations are expressed as a function of the sur-face jamming limit qi

max and the fraction of filled latticesites θ*:

where R is the average number of binding sites coveredby a bound CBM2a molecule, and B1 and B2 are constants

∂c∂t

) DL∂

2c∂z2

- u∂c∂z

-(1 - ε)

ε

∂s∂t

(1)

s(r,z,t) ) âci(r,z,t) + Fpqi(r,z,t) (2)

No ) Dp|∂ci

∂r |r)R) kf(c -

ci

â|r)R

) (3)

∂ci

∂t)

Dp

r2∂

∂r(r2∂ci

∂r) -Fp

â∂qi

∂t(4)

1qi

max

∂qi

∂t) µfci(1 - θ*)[1 - B1(R)θ* - B2(R)(θ*)2]2 -

µrθ* (5)

Biotechnol. Prog., 2004, Vol. 20, No. 5 1481

that depend on R in the following ways:

Under local equilibrium conditions, eq 5 reduces to

where Ka is the equilibrium association constant (M-1)given by µf/µr.

Boundary Conditions and Solution Algorithm.Dankwerts’ (22) initial and boundary conditions wereapplied to the solution of the one-dimensional continuityequation (1) applied to the target protein. The comple-mentary set of initial and boundary conditions for eq 4,describing solute diffusion and binding within the sta-tionary-phase particle volume, are

Equation 3 provides the final spatial boundary condi-tion by specifying that the concentrations ci and c in theparticle volume at r ) R and in the mobile-phaseinterstitial space, respectively, are coupled by the rateof mass transfer through the fluid film.

Numerical solution of eqs 1-10 was achieved using afinite-difference iteration scheme. Spatial derivatives inall equations were discretized using the control-volumemethod of Patankar (23), yielding a set of nonlinearreaction-diffusion equations that were solved by a New-ton-Raphson root-finding method (24). A second-orderAdams-Bashforth predictor-corrector method (25) wasused to perform the time integration.

Results and DiscussionAvicel PH101 Affinity Column Parameters and

Properties. Table 1 provides measured parameters forthe packed Avicel PH101 affinity columns used in thesestudies. The column diameter to particle diameter ratioof 172 is well above the minimum value required to safelyignore additional dispersion effects that may occur at thecolumn wall (26). Evidence that the Avicel PH101 ispacked uniformly within the column is provided by twoexperimental measures of column packing efficiency, thereduced plate height h and the peak asymmetry factorAs. For an efficiently packed column, the reduced plateheight should be close to 2 (27) and the asymmetry factorshould be close to unity.

The column void fraction ε at relatively low backpressures was regressed from elution data for pulseinjections of ferritin (MW ) 440 kDa). Elution peaks forthis nonpenetrating nonbinding protein were Gaussianor very nearly Gaussian in shape, allowing the firstmoment µ1 to be equated to the peak retention time tRaccording to the theory of Haynes and Sarma (28):

In accordance with eq 11, Figure 1 shows first momentdata for ferritin pulses as a function of u over the range6.37 × 10-3 to 2.54 × 10-2 cm s-1.

Pressure drop across the column was also monitoredas a function of u. Application of the Blake, Kozeny, andCarmen (29) equation

to column pressure drop (∆P, Pa) data yields a columnresistance factor, defined as 36k(1 - ε)2/e3, of 660 ( 30.For spherical packing, the aspect factor k is assumedequal to 5 (30). The regressed column resistance factortherefore corresponds to a column void fraction of0.44 ( 0.03, which is in close agreement with theindependently measured value reported in Table 1,indicating that the column packing density is reasonablyinsensitive to back pressures generated at higher flowrates.

Avicel PH101 Pore-Size Distribution. The distribu-tion of pore sizes in fully hydrated Avicel PH101 particleswas determined by the solute exclusion method of Lin etal. (31) using poly(ethylene glycol) standards of knownweight-average molecular weight and hydrodynamic

B1(R) ) 0.7126R + 1.404R1.5

1 + 3.4363R + 2.4653R1.5(6)

B2(R) ) 0.07362R + 0.1204R1.5

1 + 0.5433R + 0.2725R1.5(7)

θ* )Kaci[1 - B1(R)θ* - B2(R)(θ*)2]2

1 + Kaci[1 - B1(R)θ* - B2(R)(θ*)2]2(8)

ci ) 0 t ) 0 0 e r e R (9)

∂ci

∂r |r)0) 0 r ) 0 t > 0 (10)

µ1 ) tR ) Lu

[ε + (1 - ε)â] (11)

Table 1. Packed Avicel PH101 Affinity ColumnParameters and Properties

column property value

temperature 20 °CL 4.7-9.5 cmD 1 cmdp 58 ( 12 µmFp 930 ( 5 kg m-3

ε 0.45 ( 0.03h (reduced plate height)a 2.4 ( 0.2As (peak asymmetry factor)b 1.08 ( 0.03

a The reduced plate height h, defined as H/dp, was calculatedfrom elution data for sodium nitrate (u ) 0.0254 cm s-1) usingthe relation h ) L/[5.54(VR/wh)2dp] originally proposed by vanDeemter et al. (39). b Calculated from the elution peak for sodiumnitrate (u ) 0.0254 cm s-1) at 10% peak height according to themethod of Hagel (50).

Figure 1. Measured first central moment (µ1) as a function ofinterstitial velocity u of the mobile phase for 100-µL pulses offerritin over the velocity range u ) 6.37 × 10-3 to 2.54 × 10-2

cm s-1. Mobile phase contained 50 mM phosphate buffer (pH 7,20 °C).

∆P ) 36kuε

Lηdp

2

(1 - ε)2

ε3

(12)


radii. Lin et al. (31) showed that PEG standards areproduced as nearly monodisperse fractions (1.02 < dp <1.05) and do not interact preferentially with cellulose,making them an ideal choice for our exclusion studies.

Figure 2 plots the porosity â of the sieved (fraction 2)Avicel PH101 particles as a function of the hydrodynamicdiameter of the probe. When the probe’s hydrodynamicdiameter exceeds 10 Å, the porosity decreases steadilyfrom its maximum of ca. 0.7. The particles are essentiallynonporous for probes or proteins with average diametersgreater than 70 Å. Determination of â for recombinantproteins containing a CBM2a tag was facilitated byfitting the data in Figure 2 to a Boltzmann-type functionof the form

where R, γ, and δ are fitted parameters and X is themolecular probe diameter in Å. The solid curve in Figure2 represents the best fit, for which R ) 0.73, γ ) 0.1423,and δ ) 4.5861.

Recombinant Expression in E. coli of the ProteinA-CBM2a Fusion. Protein production and final celldensities in batch fermentations of recombinant E. coliare often inhibited by high levels of organic acidsproduced when the culture is subjected to high concen-trations of glucose. Fed-batch fermentations, where es-sential nutrients including glucose are periodically fedat noninhibiting levels, generally yield significantlyhigher final cell densities and target protein titers (18,32, 33).

Figure 3 shows typical growth and protein A-CBM2aproduction curves for a fed-batch fermentation of recom-binant E. coli JM101 harboring the pSACBM2a plasmidas described earlier. The arrows in Figure 3 indicatewhere glucose additions to a final concentration of 2 gL-1 were made. Final cell densities between 6 and 7optical density (600 nm) units were generally achieved,resulting in protein A-CBM2a supernatant concentra-tions between 200 and 250 mg L-1. Glucose concentra-tions remained low throughout the culture, and a directcorrelation between cell density and protein productionwas observed.

The final culture supernatant containing proteinA-CBM2a was separated from the cells by centrifugationat 10,000 rpm for 10 min. Lane 2 of the 15% SDS-PAGE

gel (Figure 4) shows that the resulting clarified super-natant is rich in protein A-CBM2a; the target fusionprotein accounts for 25-30% of the total protein content.A single 42-kDa protein, which corresponds to the mo-lecular weight of protein A-CBM2a, is shown to elute(lane 4) from the Avicel PH101 column following columnwashing (lane 3), indicating that the chromatographicmedia is highly specific for CBM2a and recombinant

Figure 2. Porosity â of sieved (fraction 2, mean particlediameter dp of 58 µm, 45 µm < dp < 75 µm) Avicel PH101particles as a function of the hydrodynamic diameter of theprobe. Curve represents best fit of eq 13, for which R ) 0.73,γ ) 0.1423, and δ ) 4.5861.

Figure 3. Growth and protein A-CBM2a production curvesfor a fed-batch fermentation of E. coli JM101 harboring thepSACBM2a plasmid. Inoculum was grown to an OD (600 nm)of 1 on M9 media and then added to 400 mL of fresh media in2-L shake flasks to culture at 37 °C for 37 h. Glucose wasperiodically added (arrows) to the culture to a final concentrationof 2 g L-1, and proline (0.25 g L-1) and kanamycin (50 µg mL-1)were added at 23 h of culture time: (O) glucose concentration(g L-1); (0) optical density; (3) protein A-CBM2a concentration(mg L-1).

Figure 4. SDS-PAGE gel documentation of the affinitypurification of protein A-CBM2a from the supernatant of a 400-mL recombinant E. coli JM101 culture. Lane 1: molecular massstandards myosin (200 kDa), â-galactosidase (116.25 kDa),phosphorylase B (97.40 kDa), bovine serum albumin (66.2 kDa),ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsininhibitor (21.5 kDa), lysozyme (14.4 kDa), aprotinin (6.5 kDa).Lane 2: culture supernatant. Lane 3: proteins eluted fromAvicel PH101 resin during column wash step. Lane 4: pooledproduct peak following elution from the 7.5 mL Avicel PH101column with pure water.

â ) R1 + exp(γX - δ)

(13)


proteins tagged with it and shows little or no nonspecificbackground binding.

One-Step Affinity Purification of Protein A-CBM2a from Culture Supernatant. Figure 5 shows atypical chromatogram for the loading, concentrated saltwash (1 M NaCl in 50 mM phosphate buffer, pH 7), dilutesalt wash (50 mM phosphate buffer, pH 7), elution, andregeneration (6 M Gu-HCl) stages of the affinity purifica-tion of protein A-CBM2a from clarified culture super-natant. A 1.027-L portion of culture supernatant con-taining 217 mg L-1 protein A-CBM2a (Co) was loadedonto the column at an interstitial velocity u of 2.12 ×10-2 cm s-1 (pH 7, 20 °C). Initial flow-through of non-binding contaminating proteins occurs in approximatelyone void volume. Sample was loaded onto the columnuntil initial breakthrough of protein A-CBM2a, mea-sured as 1% of Co, was observed after ca. 510 min ofcontinuous loading based on antibody-based dot-blotanalysis of 1-mL eluent fractions.

Bound protein A-CBM2a was eluted from the washedcolumn with pure water, suggesting that the affinitycolumn behaves much like a highly selective hydrophobicinteraction chromatography (HIC) column. Competitionisotherm studies (13) show that CBM2a specifically bindsto crystalline surfaces of insoluble cellulose. Binding isdriven by the entropy increase accompanying dehydra-tion of the contacting protein and sorbent surfaces (14).This dehydration driving force is minimized in purewater, allowing most fusion proteins containing a CBM2atag to be eluted (34). As reported in Table 2, the purity(based on densitometry analysis of lane 4 of Figure 4)and product concentration factor in the pooled productelution peak are comparable to those typically reportedfor other fusion-tag-based affinity chromatography sys-tems. The average product yield of 67%, however, issomewhat lower than is generally reported for purifica-tion of polyhistidine-tagged proteins using IMAC (35, 36).As shown in Figure 5, the remaining 33% of boundprotein A-CBM2a is quantitatively recovered in the

6 M GuHCl regeneration step, making the technologyparticularly useful for production and purification ofprotein fusions for which robust refolding protocols havebeen established.

Determination of Model Parameters. Because ofthe availability of a large stock of purified CBM2a, initialmodel development and validation were based on frontalchromatography experiments using aqueous CBM2asolutions as feed. Once validated, the model was thenapplied to the prediction of protein A-CBM2a break-through during frontal loading of clarified supernatantfrom a recombinant E. coli JM101 culture harboring thepSACBM2a plasmid encoding protein A-CBM2a.

Table 3 provides a listing of measured and calculatedparameters describing geometric, transport, and ther-modynamic properties of the packed Avicel PH101 affin-ity column for the case where either CBM2a or proteinA-CBM2a is the target protein to be purified. Theeffective resin porosity for each protein was calculatedusing eq 13. The equilibrium binding constant Ka andthe surface jamming limit (i.e., the maximum bindingcapacity) qi

max of the Avicel PH101 affinity matrix wereobtained by fitting eq 8 to equilibrium binding isothermdata, such as that shown in Figure 6 for CBM2a. Thebulk diffusivity Dm (m2/s) of each protein in water wasestimated using the Polson equation (37) and the refinedcorrelation parameters of Tyn and Gusek (38).

The remaining transport parameters were determinedby regression of the equivalent plate height (HETP)theory of van Deemter et al. (39) to measured elutionchromatograms for nonbinding noninteracting solutes ofsimilar size and shape to CBM2a and protein A-CBM2a.For Gaussian-shaped elution peak profiles, the secondcentral moment µ2 is equal to the standard peak varianceσ2. In accordance with the original theory of van Deemteret al. (39), Ruthven (40) and others (28) have shown fora solute under nonbinding conditions that one HETP isequal to

Figure 5. Typical chromatogram for the (1) loading, (2)concentrated salt wash (1 M NaCl in 50 mM phosphate buffer,pH 7), (3) dilute salt wash (50 mM phosphate buffer, pH 7), (4)elution in pure water, and (5) regeneration (6 M Gu-HCl) stagesof the affinity purification of protein A-CBM2a from clarifiedculture supernatant; 1.027 L of culture supernatant containing217 mg L-1 protein A-CBM2a (Co) was loaded onto the columnat an interstitial velocity u of 2.12 × 10-2 cm s-1 (pH 7, 20 °C).

Table 2. Analysis of Performance of One-Step AffinityPurification of Protein A-CBM2a

column property value

purity >95%yield 67 ( 3%Ce/Co (concentration factor) 34 ( 3As

a 1.11 ( 0.04a Calculated from the product elution peak at 10% peak height

according to the method of Hagel (50).

Table 3. Measured or Calculated Properties andParameters Describing the Packed Avicel PH101 Columnwith Either CBM2a or Protein A-CBM2a as the TargetProtein

target protein

parameter CBM2a protein A-CBM2a units

â 0.45 0.10Ka 9500 ( 50 9470 ( 60 m3/molqi

max (surfacejamming limit)

2.86 ( 0.05 0.66 ( 0.03 mol/m3

Dl 8.0 ( 2.4 × 10-8 5.2 ( 2.1 × 10-8 m2/skf 7.83 ( 0.1 × 10-6 5.1 ( 0.1 × 10-6 m/sDm 12.8 × 10-11 9.9 × 10-11 m2/sDp 2.2 ( 0.2 × 10-12 1.6 ( 0.2 × 10-12 m2/sµf g0.4a g0.4 m3/mol/sPe 2900-21000 3700-24000Bi 34 ( 4 31 ( 3Da g153 ( 6 g210 ( 6

a Data taken from Jervis (44).


where tr is the average retention time (s) of the solute inthe column, uo is the superficial fluid velocity, and

Equation 14 can be linearized with respect to uo2

to allow for determination of the axial dispersion coef-ficient Dl and the overall mass transfer coefficient Kmfrom the intercept and slope, respectively, of a plot ofuoxHETP as a function of uo

2.Horse heart myoglobin (MW ) 17,800 Da) is a globular

protein similar in size and shape to CBM2a, which showsno binding affinity for Avicel PH101. We have thereforeassumed that the transport properties of CBM2a in ourAvicel PH101 affinity column are equivalent to those formyoglobin in the same column. Figure 7 plots measuredHETP data for 200-µL 15.8-µM pulses of myoglobin (in50 mM phosphate buffer, pH 7) loaded onto a uniformlypacked 14.2 cm3 column of sieved Avicel PH101 at 20 °Cand superficial velocities ranging from 6.37 × 10-5 to2.54 × 10-4 m s-1. Elution peaks were Gaussian withminimal peak tailing at all superficial velocities studied.Regression of the linear data set (R ) 0.9918) to eq 16gives values for Dl and Km of 8.0 ( 2.4 × 10-8 m2 s-1 and4.62 ( 0.23 × 10-2 s-1, respectively.

Segregation of the overall mass transfer coefficient Kminto its film and intrapore mass transfer contributionsrequires an appropriate correlation for estimating thevalue of either kf or Dp. For porous spherical particlessuch as Avicel PH101, good correlations for estimatingthe film mass-transfer coefficient kf are available (41, 42).Foo and Rice (43), for instance, proposed the followingdimensionless correlation for low Reynolds number flows

where Sh is the Sherwood number and Re and Sc arethe Reynolds and Schmidt numbers, respectively, givenby

Equations 17 and 18, where ν is the liquid kinematicviscosity (m2 s-1), were used to determine the estimatedvalue of kf reported in Table 3. Estimates of kf for CBM2afrom the correlations of Goto et al. (42) and Wakao et al.(41) differed from that reported by less than 10%.

Using time-resolved confocal microscope images forFITC-labeled CBM2a, Jervis (44) estimated a forwardrate constant of 0.4 m3 mol-1 s-1 for CBM2a binding tothe surface of 1.2-µm thick sheets of crystalline celluloseproduced by Valonia ventricosa. This represents a lowerlimit on the true value of the intrinsic adsorption rateconstant µf as mass transfer resistances in the fluid filmwere not considered in the analysis.

Model Predictions for CBM2a. The particle Peclet(Pe ) 2Rpν/kf), Biot (Bi ) kfRp/3Dp), and Damkohler(Da ) µfRp

2/Dp) numbers for processing of CBM2a on ourAvicel PH101 affinity column (Table 3) indicate thatsolute dispersion within the column is largely controlledby intrapore diffusion with appreciably smaller but notinsignificant contributions from mass transfer. Moreover,the large value of the Damkohler number indicates thatsolute binding is rapid compared to pore diffusion,allowing the local equilibrium assumption to be appliedthroughout the column and local sorbate concentrationsto be calculated using eq 8. Intrapore diffusion is oftenthe rate-limiting process in column chromatographyinvolving porous resins (1, 45). Furthermore, Hall et al.(46) have shown that the assumption of local solute-sorbate equilibria is exact when Co, the concentration ofsolute in the feed, is significantly greater than 1/Ka, whichis true in all cases reported here. All model calculationsare therefore based on finite-difference solution of eqs1-4 with local sorbate concentrations calculated usingeq 8, the random-sequential-adsorption (RSA) isothermequation. Contributions to peak spreading from both

Figure 6. Adsorption isotherm (bound protein concentrationqi versus free protein concentration in the pore liquid ci) forbinding of CBM2a to Avicel PH101 at 20°C and pH 7 (50 mMphosphate buffer). Curve represents best fit of eq 8 to theexperimental data set.

HETP ) σ2Ltr2

)2Dl

uo+ 2uo

1Km

( ε

1 - ε)[1 + ε

(1 - ε)â]-2

(14)

1Km

)Rp

2

15âDp+

Rp

3kf(15)

uoxHETP )uoσ

2L

tr2

)

2Dl + 2uo2 1Km

( ε

1 - ε)[1 + ε

(1 - ε)â]-2(16)

Figure 7. Height-equivalent of a theoretical plate (HETP)values for 200-µL 15.8-µM pulses of myoglobin (in 50 mMphosphate buffer, pH 7) loaded onto a uniformly packed 14.2cm3 column of sieved Avicel PH101 at 20 °C and superficialvelocities (uo) ranging from 6.37 × 10-5 to 2.54 × 10-4 m s-1.Elution peaks were Gaussian with minimal peak tailing at allsuperficial velocities studied.

Sh )2Rpkf

Dm) 2 + 1.45Re1/2Sc1/3 (17)

Re )2Rpu

νand Sc ) ν

Dm(18)


mass transfer and intrapore diffusion are explicitlyconsidered in this model, in accordance with the rela-tively low values for the estimated column Biot numbersfor both target proteins (CBM2a and protein A-CBM2a).CBM2a has an average diameter of 27 Å, which is slightlyless than half the average diameter of the pores withinAvicel PH101. As a result, approximately 40% of the porevolume within the stationary phase is inaccessible toCBM2a. For the fusion protein, protein A-CBM2a, only10% of the pore volume is accessible. As a result, boundamounts within and at the exterior surface of Avicelparticles are comparable at equilibrium.

Figure 8 shows an experimental breakthrough curvefor a 43.8 × 10-3 mol m-3 feed of pure CBM2a flowingthrough a 14.2-cm3 column of Avicel at a flowrate of 1mL min-1 (20 °C, pH 7). The curve, which is consistentwith measured breakthrough curves at all column oper-ating conditions, shows a modest but significant asym-metry such that the leading edge of the breakthroughtransition is sharper than the approach to saturation.Regression of the overlap parameter R (which specifiesthe average number of binding sites covered by a boundCBM2a molecule) in eq 8 to the breakthrough curve inFigure 8 gives an optimized value for R of 10.0 and a goodmodel fit of the data. The effect of the RSA model (eq 8)on the calculated breakthrough curve is the predictionof band broadening near column saturation conditions,as observed experimentally. This effect is not observedwhen the classic Langmuir adsorption isotherm modelis used in place of eq 8. In reality, the distortion observedin the breakthrough curve symmetry can be due to anumber of effects, including nonuniform mixing in someregions of the bed, nonspecific adsorption, and confor-mational changes in the adsorbed protein (47). Thus, itmay be more accurate to view the overlap factor R as anadjustable parameter that corrects for breakthroughcurve distortion.

Changes in feed concentration of the adsorbing soluteprovide a fairly stringent test of our column model sinceconcentration gradients in the mobile phase and acrossthe fluid film dictate the driving force toward adsorptionequilibrium. Figure 9 shows measured breakthroughcurves for frontal loading of CBM2a as a function of feedconcentration. Increasing the feed concentration de-creases elution time and increases the sharpness of the

breakthrough curve as would be expected for a self-sharpening function where pore diffusion and mass-transfer events are balanced. At all feed conditions, thebreakthrough curves are relatively sharp, indicatinguniform flow and binding characteristics.

Model calculations at each feed condition are alsoshown in Figure 9. In all cases, the model accuratelypredicts the average time of breakthrough and closelymirrors the shape of the breakthrough curve. There is asmall departure from experiment when saturation isapproached, indicating that in addition to binding-site-overlap phenomena, nonequilibrium effects such as non-uniform flow may contribute to breakthrough curveasymmetry.

Figure 10 compares model calculations with experi-mental breakthrough curves for two different columnlengths. Agreement is good, particularly up to themidpoint of the breakthrough curve. Similarly, the modelis able to predict with reasonable accuracy the influenceof mobile-phase interstitial velocity on the shape and timeof breakthrough.

Figure 8. Experimental breakthrough curve (CBM2a concen-tration c in the eluent versus elution time t) for a 43.8 × 10-3

mol m-3 feed of pure CBM2a flowing through a 14.2 cm3 columnof sieved Avicel at a flowrate of 1 mL min-1 (20 °C, pH 7). Thecurve represents the model fit where the overlap parameter Rin eq 8 has been regressed to an optimized value of 10.0.

Figure 9. Measured and predicted breakthrough curves on a7.5-mL sieved Avicel PH101 column for frontal loading ofCBM2a at 1 mL min-1 as a function of feed concentration. PureCBM2a feed concentrations for breakthrough curves from fromleft to right were 48.2 × 10-3, 28.6 × 10-3, 24.5 × 10-3, 21.0 ×10-3, and 17.3 × 10-3 mol m-3.

Figure 10. Measured and predicted breakthrough curves on asieved Avicel PH101 column for frontal loading of 28.6 × 10-3

mol m-3 CBM2a at 1 mL min-1 as a function of column length.Experimental data: (O) 4.7-cm column, (0) 6.7-cm column.Model predictions: (1) 4.7-cm column, (2) 6.7-cm column, (3)8.7-cm column, (4) 10.7-cm column, and (5) 20.7-cm column.


Model Predictions for Protein A-CBM2a. Thecolumn model derived earlier strictly applies to affinityadsorption of a single solute from a binary aqueous feedsolution. The potential effects of contaminating soluteson the binding and transport behavior of the targetprotein are not considered. Although extension of themodel to specifically include a mixture of nonbindingcontaminants is theoretically possible, our objective is toestablish a useful, simple algorithm for estimatingproduct breakthrough. To achieve this goal, we haveassumed that the contaminating solutes are sufficientlydilute that their presence does not influence the transportand binding properties of the target protein. The feedsolution can then be regarded as a pseudobinary mixtureof the fusion protein in the solvent, and the modelpresented earlier can be applied.

Modeling of breakthrough of protein A-CBM2a duringfrontal loading of a clarified recombinant cell lysaterequires target-protein specific values for Ka, qi

max, Dl,kf, and Dp. However, one often does not have sufficientpure protein available to independently measure theseparameters according to the methods described earlier.In such cases, reasonable methods are required forestimating these model parameters. To simplify modeluse, we have applied the following strategy. We assumethat the CBM2a affinity tag acts independently of thefusion partner, so that Ka for the protein A-CBM2afusion protein can be set equal to the value measuredfor CBM2a. In simulations of CBM2a elution, axialdispersion makes a relatively small contribution tobreakthrough behavior, allowing Dl for the fusion proteinto be set equal to the measured Dl for CBM2a withoutsignificant error. Finally, we assume that sufficientpurified protein is available to make a single-point batchmeasurement of qi

max (Table 3). These assumption re-duce the number of parameters that must be estimatedfor protein A-CBM2a to two: kf and Dp.

Equation 17 has been used to estimate kf by assumingthat the bulk-phase diffusivity of protein A-CBM2a canbe determined using the modified Polson equation pro-posed by Tyn and Gusek (38):

where A is a constant of value 2.85 × 10-5 cm2 s-1 g1/3

mol-1/3 and MW is the molecular weight of the protein(g mol-1). Epstein (48) has shown that the intraporediffusivity Dp can also be related to the bulk diffusivityDm through the introduction of a tortuosity factor κ thatdepends only on the properties of porous sorbent:

Application of eq 20 to the measured diffusion coef-ficient data for CBM2a gives an estimate for κ of 2.51.Epstein (48) and others (49) have predicted that forporous solids κ usually lies between 2 and 6.

Figure 11 compares predicted and measured proteinA-CBM2 breakthrough curves for a frontal load of 1 Lof culture supernatant containing 51.7 ( 17.3 µM proteinA-CBM2a entering the 7.5-mL packed Avicel column ata superficial velocity u of 2.12 × 10-4 m s-1. Modelpredictions are in excellent agreement with experiment,indicating that both the fusion-protein parameter esti-mation methods and the pseudobinary solution assump-

tion (whereby we ignore the effects of contaminatingsolutes on the binding and transport properties of proteinA-CBM2a) are reasonably accurate for the case wherethe total protein load in the feed is less than 1 g L-1.However, this modeling approach must be applied toadditional CBM2a-tagged fusion proteins to verify itsgenerality and full range of utility.

Conclusions

With the emergence of off-patent drug manufacturersand shrinking federal health budgets, production ef-ficiency and economics are becoming critical issues inbiotechnology. As a result, industry is seeking newmethods for streamlining downstream processing ofrecombinant protein products. We have demonstratedthat the CBM2a affinity tag coupled with an extremelyinexpensive cellulose-based chromatography resin (AvicelPH101) can be successfully applied to the one-steppurification of a S. aureus protein A-CBM2a fusionprotein from the supernatant of a recombinant E. coliculture. Elution chromatograms from the Avicel PH101affinity column are characterized by steep initial productbreakthrough, indicating fast binding kinetics and uni-form flow distribution, and narrow elution peaks, allow-ing for the recovery of a concentrated protein product ofhigh purity. A one-dimensional differential column modelincorporating a random-sequential-adsorption isothermmodel and a pseudobinary-solution approximation wasshown to provide accurate predictions of product elutionover a range of column operating conditions and scales.A set of useful correlations was established to obtaincritical model parameters for fusion proteins with aCBM2a affinity tag. Although further testing is required,the model appears useful for optimizing column operatingconditions and feed cycles for the affinity purification ofany protein fused to the CBM2a affinity tag.

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(19)

Dp )âDm

κ(20)

Figure 11. Measured and predicted protein A-CBM2 break-through curves for a frontal load of 1 L of culture supernatantcontaining 51.7 ( 17.3 µM protein A-CBM2a entering the 7.5-mL sieved Avicel PH101 affinity chromatography column at asuperficial velocity u of 2.12 × 10-4 m s-1.


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Accepted for publication May 18, 2004.

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