Engineering Soluble Monomeric Streptavidin with Reversible BiotinBinding Capability*□S

Received for publication, February 15, 2005, and in revised form, April 12, 2005Published, JBC Papers in Press, April 19, 2005, DOI 10.1074/jbc.M501733200

Sau-Ching Wu and Sui-Lam Wong‡

From the Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Monomeric streptavidin with reversible biotin bind-ing capability has many potential applications. Becausea complete biotin binding site in each streptavidin sub-unit requires the contribution of tryptophan 120 from aneighboring subunit, monomerization of the natural tet-rameric streptavidin can generate streptavidin with re-duced biotin binding affinity. Three residues, valine 55,threonine 76, and valine 125, were changed to eitherarginine or threonine to create electrostatic repulsionand steric hindrance at the interfaces. The double mu-tation (T76R,V125R) was highly effective to monomerizestreptavidin. Because interfacial hydrophobic residuesare exposed to solvent once tetrameric streptavidin isconverted to the monomeric state, a quadruple mutein(T76R,V125R,V55T,L109T) was developed. The first twomutations are for monomerization, whereas the last twomutations aim to improve hydrophilicity at the inter-face to minimize aggregation. Monomerization was con-firmed by four different approaches including gel filtra-tion, dynamic light scattering, sensitivity to proteinaseK, and chemical cross-linking. The quadruple muteinremained in the monomeric state at a concentrationgreater than 2 mg/ml. Its kinetic parameters for interac-tion with biotin suggest excellent reversible biotin bind-ing capability, which enables the mutein to be easilypurified on the biotin-agarose matrix. Another mutein(D61A,W120K) was developed based on two mutationsthat have been shown to be effective in monomerizingavidin. This streptavidin mutein was oligomeric in na-ture. This illustrates the importance in selecting theappropriate residues and approaches for effective mo-nomerization of streptavidin.

(Strept)avidin with reversible biotin binding capability canextend the applications of the biotin-(strept)avidin technology.These molecules can be applied for affinity purification of bi-otinylated biomolecules, screening of ultratight binders bind-ing to biotinylated biomolecules displayed on the phage displaysystem, and development of reusable biosensor chips, protein/antibody microarrays, and enzyme bioreactors (1, 2). (Strept)a-vidin is a homotetrameric molecule with a biotin binding site in

each subunit (3). The three-dimensional structure of (strept)a-vidin (4, 5) suggests that a complete biotin binding pocket ineach subunit requires the contribution of a tryptophan residuefrom an adjacent subunit. Site-directed mutagenesis studiesalso demonstrate the importance of this residue for tight biotinbinding and subunit communications (6–8). Therefore, devel-opment of monomeric (strept)avidin can be an attractive ap-proach to engineer (strept)avidin with reversible biotinbinding capability.

The engineering of (strept)avidin to its monomeric form istechnically challenging. In the case of avidin, the first genera-tion of engineered monomeric avidin can exist in the mono-meric state only in the absence of biotin (9). This problem hasbeen solved by the recent development of the second generationof monomeric avidin (10), which carries two mutations(N54A,W110K). Structural alignment of avidin and streptavi-din indicates that these two residues correspond to Asp-61 andTrp-120 in streptavidin (11). However, a streptavidin muteindesignated AK,1 which carries the corresponding double muta-tions (D61A,W120K), does not become monomeric as demon-strated in the present study. This illustrates the need to iden-tify a new set of critical residues in combination with effectiveapproaches to generate monomeric streptavidin with minimalmutational changes. Development of monomeric streptavidinhas been reported previously through mutation of three resi-dues to alanine (12). However, the low affinity of this muteintoward biotin (Kd � 1.7 � 10�6 M) makes it less than ideal formany applications.

To develop better versions of monomeric streptavidin, threeresidues (Val-55, Thr-76, and Val-125) were selected for site-directed mutagenesis. In combination with L109T mutation, aseries of single, double, and quadruple streptavidin muteinswere created and produced from Bacillus subtilis via secretion.As they were produced in the soluble form without the require-ment of refolding (13, 14), their oligomeric state can be rapidlyanalyzed by SDS-PAGE using culture supernatants from thestreptavidin mutein production strains. The muteins were pu-rified and further characterized by different approaches toconfirm their monomeric state. Their kinetic parameters forbiotin binding were determined by surface plasmon resonance-based biosensor studies.

EXPERIMENTAL PROCEDURES

Construction of Streptavidin Mutants—Different point mutationswere introduced to the coding sequence of a synthetic streptavidin gene(ssav) in the B. subtilis expression vector pSSAV-Tcry (14) by PCR-

* This work was supported by a discovery grant from the NaturalSciences and Engineering Research Council of Canada and a short termproject grant from the University of Calgary. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Supplemental Figs. S1, S2, and S3.

‡ To whom correspondence should be addressed: Dept. of BiologicalSciences, Division of Cellular, Molecular and Microbial Biology, Uni-versity of Calgary, 2500 University Dr., N. W. Calgary, Alberta T2N1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong@ucalgary.ca.

1 The abbreviations used are: AK, streptavidin mutein with doublemutations (D61A,W120K); ka, on rate; kd, off rate; Kd, dissocia-tion constant (kd/ka); M1, streptavidin mutein with a single muta-tion (T76R); M2, streptavidin mutein with double mutations(T76R,V125R); M4, streptavidin mutein with four mutations(T76R,V125T,V55T,L109T); PBS, phosphate-buffered saline; sulfo-EGS, ethylene glycol bis(sulfosuccinimidyl succinate).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 24, Issue of June 17, pp. 23225–23231, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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http://www.jbc.org/cgi/content/full/M501733200/DC1Supplemental Material can be found at:

based oligonucleotide-directed mutagenesis. Five mutants (V125R,V125T, V55R, V55T, and T76R), each bearing a single mutation thatresults in the change of an amino acid residue as the name suggests,were constructed using pSSAV-Tcry as the template and the primerslisted in Table I. The amplified products were digested with the pair ofenzymes listed in Table I and cloned into pSSAV-Tcry. Five plasmids(pV125R, pV125T, pV55R, pV55T, and pT76R) resulted.

Two double mutants (M2 and AK) were also constructed. For M2(T76R,V125R), a ScaI/NheI-digested fragment of pV125R was used toreplace the corresponding fragment in pT76R. For AK (D61A,W120K),the fragment bearing the two mutations was amplified by PCR usingthe primers SAVD61AF and SAVW120KB (Table I) and the templatepSSAV-Tcry. The amplified fragment was digested by XbaI/ScaI andused to replace the corresponding fragment in pSSAV-Tcry.

The construction of M4 (T76R,V125R,V55T,L109T) involved twosteps (Supplemental Fig. S1). First, a 164-bp fragment bearing twomutations (T76R,L109T) was amplified using SAVT76RF andSAVL109TB (Table I) as primers and pSSAV-Tcry as template. Theamplified product was digested by BamHI/ScaI and used to replace thecorresponding fragment in pV55T to generate pV55T-T76R-L109T. Inthe second step, a ScaI/NheI-digested fragment of pV125R was used toreplace the corresponding fragment in pV55T-T76R-L109T togenerate pV55T-T76R-L109T-V125R.

Production and Purification of Streptavidin—Wild-type streptavidinwas produced by B. subtilis WB800(pSSAV-Tcry) cultured in a definedmedium (14). The secreted protein was purified to homogeneity usingcation exchange followed by iminobiotin affinity chromatography (12).Production and purification of streptavidin muteins followed a similarscheme with two major modifications: super-rich medium (15) was usedin place of the defined medium, and biotin-agarose (Sigma) was used inplace of iminobiotin-agarose as the affinity matrix. Dialyzed samplecontaining partially purified mutein was loaded to a 1-ml biotin-agarosecolumn. Streptavidin muteins were eluted from the column using 20mM D-biotin in phosphate-buffered saline (PBS; 50 mM sodium phos-phate, 100 mM NaCl, pH 7.2). Concentration of purified streptavidinwas determined spectrophotometrically using the known extinctioncoefficient at 280 nm (16, 17) for each individual mutein.

Determination of the Molecular Size of Streptavidin—Molecularmass of purified streptavidin and its muteins was estimated by both gelfiltration and dynamic light scattering studies. Gel filtration was per-formed on the Bio-Rad biologic work station equipped with a Bio-PrepSE 100/17 column that had been calibrated with molecular mass pro-tein markers (Bio-Rad). Molecular mass was also estimated from thehydrodynamic radius of the mutein obtained using a DynaPro MSdynamic light scattering instrument (Protein Solutions) that had beencalibrated with lysozyme. Protein samples (2–3 mg/ml in PBS) werepassed through a 0.02-�m filter (Whatman Anodisc 13) immediatelyprior to measurement. The size distribution profile was analyzed usingthe manufacturer’s Dynamics V6 software.

Proteinase K Digestion of Streptavidin and Its Muteins—Purifiedstreptavidin and its muteins (30 �M monomer) were treated with pro-teinase K (Invitrogen, 5 �M) for 15 min at 30 °C in 50 mM Tris-HCl

containing 5 mM CaCl2, pH 8.0. The reaction was stopped by precipita-tion with trichloroacetic acid (18). Boiled samples of precipitated pro-teins were resolved by reducing SDS-PAGE. The same analysis wasperformed with streptavidin samples treated with biotin (1 mM finalconcentration) prior to proteinase K digestion.

Cross-linking Reactions—Cross-linking of streptavidin and its mu-teins was carried out using ethylene glycol bis(sulfosuccinimidyl succi-nate) (sulfo-EGS) (Pierce) as the cross-linker. A typical reaction mixture(20 �l) contained the purified mutein (0.25 mg/ml) and sulfo-EGS (10-fold molar excess over the protein) in PBS. After 30 min at roomtemperature, the reaction was quenched with Tris-HCl (30 mM, pH 7.5).Aliquots of the cross-linking reaction samples were boiled and exam-ined by SDS-PAGE. Lysozyme (Sigma, 0.25 mg/ml) was included in thestudy to help establish the optimal reaction conditions.

Kinetic Analysis of Streptavidin Muteins—The kinetic parameters(both on and off rates for interaction with biotin) of streptavidin mu-teins were determined in real time using the surface plasmon reso-nance-based BIAcoreX biosensor. Biotin-conjugated bovine serum albu-min immobilized on a CM5 sensor chip was used to study thereversibility of biotin binding (12).

Computer Programs for Streptavidin Analyses—Swiss-pdb Viewer(19) was used to display streptavidin (Protein Data Bank code 1SWE(20)), analyze interfacial residues, measure distance between residues,and align the structures of streptavidin and avidin. Interfacial contactareas were calculated using the protein-protein interaction server (21)and the Formiga module in the Sting Millennium Suite (22). The plotsof accessible surface area of individual residues in streptavidin in eitherthe monomeric or tetrameric state were generated using the ProteinDossier module in the Sting Millennium Suite.

RESULTS

Selection of Key Residues in Streptavidin for Site-directedMutagenesis—Tetrameric streptavidin is arranged as a dimerof dimers (Fig. 1A). The interface between subunits A and B(and between C and D) has the most extensive subunit inter-actions. The interfacial contact area between A and B is �1,557Å2 with 17 H-bonding interactions, two salt bridges, and nu-merous van der Waals interactions. The interface contact be-tween A and D is also extensive with a contact area of 525 Å2

and two interfacial H-bonding interactions. The weakest inter-face interaction is between subunits A and C with an interfa-cial contact area of 171 Å2. To engineer monomeric streptavidinwith a minimal number of mutated residues, an attractiveapproach is to introduce both charge repulsion and steric hin-drance at these interfaces. As protein has structural plasticity(23–25), it is vital to select interfacial residues located on arigid surface to maximize the effects of charge repulsion andsteric hindrance. Because streptavidin subunit forms an eight-antiparallel stranded �-barrel structure (4, 5), the selected

TABLE IMutagenic primers for the construction of streptavidin mutants

Mutated codons are bolded and underlined. Restriction enzymes in parentheses after the mutants refer to the set of enzymes used for cloning.

V125R,V125T (ScaI/SphI)Forward primer SAVV125RTF

5�-GGAAAAGTACTCTTA(C/G)AGGACATGATACATTTAC-3�Backward primer pUB18H3

5�-GATTTCATACACGGTGCCTG-3�V55R,V55T (XbaI/SphI)

Forward primer SAVV55RTF5�-GAATCTAGATACA(C/G)ACTTACAGGAAGATATG-3�

Backward primer pUB18H3T76R (BamHI/SphI)

Forward primer SAVT76RF5�-GTGGATCCGGAACAGCACTTGGATGGAGAGTT-3�

Backward primer pUB18H3D61A,W120K (XbaI/ScaI)

Forward primer SAVD61AF5�-CATCTAGATACGTGCTTACAGGAAGATATGCATCTGCACCT-3�

Backward primer SAVW120KB5�-CAAGAGTACTTTTTTTTGCATTTGCTTC-3�

T76R,L109T (BamHI/ScaI)Forward primer SAVT76RFBackward primer SAVL109TB

5�-GAGAGTACTTTTCCATGCATTTGCTTCTGTTGTTCCAGATGTTAATGTCCATTGTGTG-3�

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residues should be located on the �-strands rather than in theloop regions. Furthermore the selected residue in one subunitshould be located very close to the equivalent residue or acharged residue in another subunit at the interface. Examina-tion of interfacial residues (Fig. 1, B–D) shows that Thr-76,Val-125, and Val-55 meet the criteria. Hence they were selectedfor mutagenesis.

Effects of Single Mutations on Monomerization of Streptavi-din—Streptavidin muteins carrying a single amino acid changeat the selected site were produced in their soluble form by B.subtilis via secretion. Analysis of non-boiled culture superna-tants by SDS-PAGE offers a quick screen for the mutationeffect (12). Weaker subunit interaction would result in a higherpercentage of the sample in the monomeric state on the SDS-polyacrylamide gel. Because biotin can strengthen subunit in-teraction, samples were analyzed in the presence or absence ofbiotin (9, 26). The impact of the mutation on weakening of thesubunit interaction followed the order: T76R � V125R �V125T � V55R � V55T (Fig. 2 and Table II). The T76R mutein(designated M1) existed 100% in the monomeric state on the

SDS-polyacrylamide gel even in the presence of biotin. In con-trast, V55T mutation had the lowest impact with the majorityof molecules in the tetrameric state even in the absence ofadded biotin. Presence of biotin shifts the majority of the threeremaining muteins (V125R, V125T, and V55R) to the tet-rameric state. As expected, changing valine to arginine exertedgreater impact than changing it to threonine. This is true forboth Val-125 and Val-55.

Effects of Multiple Mutations on Monomerization of Strepta-vidin—To develop idealized monomeric streptavidin muteinsthat are more likely to remain in the monomeric state at highstreptavidin concentrations and have excellent reversible bio-tin binding capability, two more muteins were created. M2 isthe double mutant carrying both the T76R and V125R muta-tions. M4 is a quadruple mutant carrying T76R, V125R, V55T,and L109T mutations. In this combination, the three interfa-cial hydrophobic residues Val-125, Val-55, and Leu-109 werechanged to hydrophilic ones. The last construct is AK, a doublemutant (D61A,W120K) carrying two mutations (equivalent tothose performed in avidin) that have been shown to convert

FIG. 1. Structure of tetrameric streptavidin and critical interface residues selected for mutagenesis. A, interfaces of tetramericstreptavidin. Subunit A forms three subunit contact interfaces with other subunits. These interfaces include A/B, A/C, and A/D. Subunits A, B, C,and D are highlighted in red, orange, yellow, and green, respectively. B, local environment of Thr-76 in subunit A showing the interfacial interactionbetween subunits A and B. Thr-76 and Arg-59 in subunit A are highlighted in red and pink, respectively. Thr-76 and Arg-59 in subunit B are indark and bright yellow, respectively. Thr-76 in subunit A is 4 Å from Thr-76 and 3.75 Å from Arg-59 in subunit B. Replacement of Thr-76 by anarginine would create both electrostatic repulsion (Arg-76 (subunit A)-Arg-76 (subunit B), Arg-76 (subunit A)-Arg-59 (subunit B), and Arg-76(subunit B)-Arg-59 (subunit A)) and steric hindrance at the interface for subunits A and B. Equivalent effects will also be generated at the interfacebetween subunits C and D. C, local environment of Val-125 in subunit A showing the interfacial interaction between subunits A and D. Val-125(red) in subunit A is 3.97 Å from Val-125 (green) in subunit D. It fits into a pocket formed by Val-125 (green) and Thr-123 (green) from subunit D.A change of Val-125 to arginine will create both charge repulsion and steric hindrance at the A/D subunit interface. The same is true for the B/Cinterface interactions. D, local environment of Val-55 in subunit A. Val-55 (red) in subunit A is 3.97 Å from Arg-59 (yellow) in subunit B.

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tetrameric avidin to the monomeric state (10). As shown in Fig.3 and Table II, just like M1, all these muteins existed inmonomeric state on the SDS-polyacrylamide gel even in thepresence of biotin.

Purification of Streptavidin Muteins—Purification of M4 wasused as an example to illustrate the process (Fig. 4). Proteinspartially purified by ion exchange chromatography (lane 2)were applied to a biotin-agarose column. M4 could be readilyeluted off from the column using biotin-containing buffer as theeluant (lanes 5–7). Pure streptavidin mutein obtained by thissimple procedure, after removal of biotin by dialysis, could beused for biochemical characterizations. To demonstrate thatdialysis could effectively remove any bound biotin from the M4mutein, the dialyzed sample was reloaded to the biotin-agarosematrix. Over 95% of the sample could be retained on the col-umn and eluted off from the column using biotin (data notshown). Of all the muteins, M1 tended to have a long trailingtail during elution. This indicates that M1 may not have thedesirable reversible biotin binding property. Therefore, it wasnot characterized further.

Determination of Apparent Molecular Mass of StreptavidinMuteins by Gel Filtration—Observation of 100% monomeriza-tion of the streptavidin mutein using a non-boiled sample forSDS-PAGE does not always truly reflect its existence in themonomeric state in solution because SDS can promote subunitdissociation (27, 28). The apparent molecular masses of thepurified wild-type streptavidin and the three muteins (M2, M4,and AK) were estimated by gel filtration (Supplemental Fig. 2Aand Table III). The expected molecular mass of monomericstreptavidin is 16.5 kDa. M2 and M4 in the absence of biotinshowed the apparent molecular masses of 19.95 and 21.87 kDa,respectively. These masses increased slightly in the presence ofbiotin. These data suggest that the muteins are monomeric innature because their masses are less than that for the strepta-vidin dimer (33 kDa). In contrast, the AK mutein showed anapparent molecular mass of 45.66 kDa even in the absence ofbiotin. This indicates the oligomeric nature of this mutein.Supplemental Fig. 2B shows the elution profile of purified M4(in the absence of biotin) from the gel filtration column. Thesample (loaded at 2 mg/ml) was eluted as a single peak. Thereis no evidence for the presence of tetrameric streptavidin,which would be eluted at 30.5 min.

Determination of Apparent Molecular Mass of StreptavidinMuteins by Dynamic Light Scattering—Because the apparentmolecular mass of wild-type streptavidin in the absence ofbiotin is 10 kDa less than expected (56 instead of 66 kDa) asdetermined by gel filtration, dynamic light scattering (29) wasused as a second method to estimate the apparent molecularmasses. The apparent molecular mass of wild-type streptavidinobtained in this way (69 kDa) was closer to that expected (66kDa) (Table III). The apparent molecular masses for both M2and M4 in the absence of biotin indicated that they were in themonomeric state. Addition of biotin caused only a slight in-crease in their apparent molecular masses. The AK muteinagain was found to be oligomeric independent of the presenceor absence of biotin.

Proteinase K Sensitivity of Streptavidin Muteins—Mono-meric streptavidin is expected to be more susceptible to pro-teinase K digestion (10). Therefore, wild-type streptavidin andits muteins were treated with proteinase K (Fig. 5A). Wild-typestreptavidin was converted to the core form independent of thepresence or absence of biotin. Under the condition used, thecore streptavidin was resistant to further degradation by pro-teinase K. In contrast, all three muteins including AK, M2, andM4 were much more susceptible to proteinase K digestion.Sensitivity to proteinase K is more apparent for M2 and M4,which were completely digested independent of the presence orabsence of biotin. This property is consistent with the mono-meric nature of these muteins. The AK mutein behaved differ-ently. Although most of it was digested by proteinase K in theabsence of biotin, it became much more resistant to proteinaseK when biotin was present.

Cross-linking of Streptavidin and Its Muteins—Tostrengthen the idea that both M2 and M4 are monomericwhereas AK is oligomeric in nature, protein cross-linking wascarried out using sulfo-EGS as the cross-linking agent. Sulfo-EGS reacts with both the accessible �-amino groups at the Ntermini and the surface-exposed �-amino groups of the lysineside chains in proteins. Secreted wild-type streptavidin haseight lysine residues in each subunit. The three-dimensionalstructural model of streptavidin suggests that lysine 121 insubunit A is 14.1 Å from lysine 121 in subunit D. As the spacerarm in sulfo-EGS is 16.1 Å, subunits A and D (same for sub-units B and C) should be easily cross-linked by sulfo-EGS. Alsoit is possible to have cross-linking between subunits A and B asthe N-terminal region from subunit A, which contains twolysine residues, is likely to be positioned close to lysine 80 insubunit B. The same is true for subunits C and D. Therefore,one should be able to differentiate tetrameric streptavidin fromthe monomeric form with the observation of cross-linked tet-rameric streptavidin using sulfo-EGS. Lysozyme, well knownto be monomeric in solution (30, 31), served as the negativecontrol. Fig. 5B shows that the amount of dimeric lysozymeincreased slightly in the presence of the cross-linking agent.This helped set the upper limit of the concentration of sulfo-EGS to be used under the experimental condition. The wild-type streptavidin subunit had an apparent molecular mass of19 kDa on the SDS gel. After treatment with sulfo-EGS, mostof these subunits were cross-linked to dimers and higher oli-gomers with small amounts remaining in the monomeric state.M2 and M4 muteins behaved very similarly (data for M2 arenot shown). The majority of the M2 and M4 muteins after thecross-linking treatment migrated as monomers with smallamounts in the dimeric form. These dimers may representcross-linked monomeric subunits that were artificially gener-ated in the same manner as with lysozyme. AK showed across-linking profile very similar to that of the wild-typestreptavidin. These data strongly support the idea that M2 and

FIG. 2. Western blot analysis of culture supernatants fromB. subtilis strains producing streptavidin muteins carrying asingle mutation. 15 �l of non-boiled sample of culture supernatantwas loaded to each lane. Samples in the left set were collected fromB. subtilis strains cultured in super-rich medium without added biotin.Samples in the right set were from culture grown in the presence ofbiotin (20 �M). The blot was probed with polyclonal antibodies againststreptavidin. M, molecular weight markers; wt, wild-type streptavidin;�ve control, culture supernatant from WB800(pWB705HM) (34) thatdid not produce any streptavidin.

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M4 muteins are monomeric, whereas the AK mutein is oligo-meric in solution.

Reversible Interaction between Streptavidin Muteins andBiotin—The on rate and off rate of the interactions betweenstreptavidin muteins and biotin were determined by surface plas-mon resonance-based BIAcore biosensor (12). As shown in TableIV (graphical plots for M4 are shown in Supplemental Fig. S3),M2, M4, and AK had their dissociation constant (Kd) in the rangeof 10�7 M. The off rates (kd) for these muteins were almost thesame, whereas the on rate (ka) for the AK mutein was slightlylower than the rest. One of the factors affecting the on rate is thediffusion coefficient (or molecular mass) of the streptavidin mol-ecule. Because AK is oligomeric in nature, this may account forthe lower on rate for this mutein-biotin interaction.

DISCUSSION

Although streptavidin and avidin have similar three-dimen-sional structures and biotin binding properties, development ofmonomeric streptavidin is much more challenging for two rea-sons. First, streptavidin has stronger subunit interfacial inter-actions than avidin (27, 28). More potent mutations are re-quired to weaken this strong interface interaction. Second,monomerization of streptavidin may result in the surface ex-posure of hydrophobic residues that normally would be buriedat the interface in tetrameric streptavidin. This can potentiallyaffect the solubility of the monomeric streptavidin and lead toreassociation of the monomers. The problem can be less dra-matic for avidin, which is a glycosylated protein with a carbo-hydrate chain in each of the avidin subunits.

Despite the challenge, our study illustrates that, by selectinga critical residue located on a rigid surface for mutagenic studyand the introduction of charge repulsion and steric hindranceat the interface, a single mutation (T76R) can be greatly effec-tive in developing monomeric streptavidin. Besides the sugges-tion from SDS-PAGE analysis, gel filtration study of the M1mutein also indicated that the majority of M1 was eluted at aposition corresponding to the monomeric form (data notshown). The main drawback for this mutein is its elution be-havior on the biotin-agarose column. The elution profile had atypical long trailing tail. Furthermore more M1 could be recov-ered by soaking the column overnight with buffer containingbiotin. This suggests that some of the M1 population havehigher affinity to the matrix.

To increase the efficiency of streptavidin monomerization,M2 mutein was developed by combining two potent mutations(T76R,V125R). Data from gel filtration study, dynamic lightscattering, sensitivity to proteinase K, and cross-linking reac-

TABLE IISummary of the mutagenic effects on weakening of subunit interactions in streptavidin muteins as reflected by the degree of monomerization of

streptavidin muteins after SDS-PAGEEstimation of the percentage of streptavidin in monomeric and tetrameric states is based on the blots showing the migration pattern of

non-boiled samples in Figs. 2 and 3.

Streptavidin muteinNo additional biotin With additional biotin

Monomer Tetramer Monomer Tetramer

%

M1 (T76R) 100 0 100 0V125R 92 8 12 88V125T 25 75 0 100V55R 20 80 10 90V55T 1 99 0 100M2 (T76R,V125R) 100 0 100 0M4 (T76R,V125R,V55T,L109T) 100 0 100 0AK 100 0 100 0

FIG. 3. Analysis of culture supernatants from B. subtilisstrains producing streptavidin muteins carrying different com-binations of mutations. All these strains were cultivated in super-rich medium supplemented with biotin (20 �M). 15 �l of non-boiledsample of culture supernatant was loaded. A, Coomassie Blue-stainedSDS-polyacrylamide gel. Bands corresponding to tetrameric and mono-meric streptavidin are marked by an asterisk and an arrowhead, re-spectively. B, Western blot probed with polyclonal antibodies againststreptavidin. M, molecular mass markers; wt, wild-type streptavidin; C,negative control.

FIG. 4. Purification of the M4 streptavidin mutein using bio-tin-agarose. M4 mutein partially purified by Macro S column chroma-tography (PPF) was loaded on a biotin-agarose column. M, molecularmass markers; FT, column flow-through; W, pooled washing fractions;EF1–EF3, eluted fractions.

TABLE IIIMolecular mass determination of wild-type (wt) streptavidin and its

muteins by gel filtration and dynamic light scatteringIn dynamic light scattering, the estimated molecular mass (M) was

calculated from the measured hydrodynamic radius (RH) using a pro-tein calibration curve. The peaks have a polydispersity below 15%.Theoretical molecular mass is estimated from the amino acid composi-tion of the mature protein (17).

SampleGel

filtrationM

Dynamic lightscattering Theoretical MRH M

kDa nm kDa kDa

wt (no biotin) 56.23 3.69 � 0.19 69 66.0 (tetramer)AK (no biotin) 45.66 3.20 � 0.23 50AK (biotin) 50.12 3.54 � 0.38 63M2 (no biotin) 19.95 1.98 � 0.24 17 16.5 (monomer)M2 (biotin) 23.44 2.09 � 0.29 18M4 (no biotin) 21.87 2.08 � 0.31 18.1 16.5 (monomer)M4 (biotin) 24.54 2.20 � 0.14 21.5

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tion confirmed the monomeric state of M2. The elution profileof this mutein from the biotin-agarose matrix had a consider-able improvement over that of M1. When a 30-min on-columnincubation period was allowed for the eluant in between frac-tion collection, about 90% of M2 could be recovered within 3column volumes of the eluant.

To ensure that the streptavidin mutein will stably remain inthe monomeric state, more mutations were introduced to M2.The exposure of the AB (or CD) interface will expose threehydrophobic residues: Val-55, Leu-109, and Val-125. In the M2mutein, Val-125 has been changed to arginine. We chose tofurther convert Val-55 and Leu-109 to threonine, a more hy-drophilic residue, to develop the M4 mutein. Conversion tothreonine instead of arginine is preferred because proteinswith high pI are known to have nonspecific interactions viaelectrostatic interactions (32, 33). The calculated pI of M2 is8.1. If both Val-55 and Leu-109 are converted to arginine, theresulting mutein will have a pI of 9.2. This may increase thechance of charge-related nonspecific interactions. The conver-sion of these residues to negatively charged residues was notconsidered because both Val-55 and Leu-109 are located on the�-strands, and negatively charged residues are relatively poor�-strand formers.

M4 mutein shares with M2 mutein many desirable featuresof an idealized monomeric streptavidin. They both exist in themonomeric state at a reasonably high protein concentration (2mg/ml or more as used in the dynamic light scattering study).Both have excellent reversible biotin binding capability as re-flected by their on rate and off rate for biotin interaction. Bothhave a moderate pI value of 8.1 so that charge-related nonspe-cific interactions will be minimal. In addition, M4 has twofeatures that make it even more attractive in practice. Mole-cules of M2 mutein tend to aggregate in solution. Filtration ofM2 through a 0.02-�m filter was essential for obtaining a goodsignal of the mutein for dynamic light scattering studies be-cause of poor signal detection caused by the presence of smallamounts of large aggregates in an unfiltered sample. On theother hand, a decent signal could at least be obtained with anunfiltered sample of similarly prepared M4. Thus, conversionof the two hydrophobic residues (Val-55 and Leu-109) to themore hydrophilic threonine residue did help minimize aggre-

gation of the mutein. Another attractive feature is that M4 hasa remarkably sharp elution profile with its purification usingbiotin-agarose. Over 95% of the mutein could be readily elutedoff from the column using just 2 column volumes of the eluant,leading to a high rate of protein recovery.

In the site-directed mutagenesis study, it is not difficult tounderstand the impact of the mutations in the following order:T76R � V125R � V55R. Analysis of the solvent accessibility ofindividual amino acid residues with tetrameric streptavidinindicates that the solvent-accessible area of Thr-76 in subunitA is zero. Its close distances to both Thr-76 and Arg-59 insubunit B and location on a rigid surface of a �-barrel structuremake it an ideal residue to be changed to arginine to achievethe maximal electrostatic repulsion and steric hindrance ef-fects at the subunit interface (Fig. 1B). The surface-accessiblearea of Val-125 in subunit A is 1.78%. It has extensive inter-actions with Leu-109, Trp-120, Thr-123, and Val-125 in sub-unit D (Fig. 1C); Leu-109 in subunit B; and Gln-107 in sub-unit C. Its conversion to arginine results in charge repulsionin subunit D and potential steric hindrance for subunits B, C,and D. As for Val-55 in subunit A, its surface accessible areais 29.7%; and it is only close to Arg-59 in subunit B at theinterface (Fig. 1D). Thus, V55R has the least impact onmonomerization.

Although AK mutein shows reversible biotin binding prop-erty and monomeric behavior on the SDS-polyacrylamide gel, itclearly exists in the oligomeric state in solution as suggested bygel filtration studies, dynamic light scattering, and cross-link-ing pattern. This study illustrates the importance of selectingcritical residues and effective approaches to achieve the maxi-mal monomerization effect on tetrameric streptavidin.

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TABLE IVKinetic parameters for the interactions between streptavidin

muteins and biotin

Protein ka kd Kd

M�1 s�1 s�1

M

M2 1.88 � 0.07 � 104 3.22 � 0.01 � 10�3 1.71 � 0.07 � 10�7

M4 1.80 � 0.04 � 104 3.39 � 0.02 � 10�3 1.87 � 0.06 � 10�7

AK 1.46 � 0.05 � 104 3.59 � 0.03 � 10�3 2.46 � 0.10 � 10�7

FIG. 5. Determination of the monomeric or oligomeric states of wild-type streptavidin and its muteins. Pictures show the CoomassieBlue-stained SDS-polyacrylamide gel. A, proteinase K digestion. B, cross-linking study using sulfo-EGS as the cross-linker. All samples were boiledprior to loading. M, molecular weight markers; wt, wild-type streptavidin; L, lysozyme. Numbering represents streptavidin molecules in monomeric(1), dimeric (2), and oligomeric (3–5) states, respectively.

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