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  • 1. Supplemental Material can be found at: http://www.jbc.org/cgi/content/full/M501733200/DC1THE 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.Engineering Soluble Monomeric Streptavidin with Reversible BiotinBinding Capability*□ S Received for publication, February 15, 2005, and in revised form, April 12, 2005 Published, 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- each subunit (3). The three-dimensional structure of (strept)a-ing capability has many potential applications. Because vidin (4, 5) suggests that a complete biotin binding pocket ina complete biotin binding site in each streptavidin sub- each subunit requires the contribution of a tryptophan residueunit requires the contribution of tryptophan 120 from a from an adjacent subunit. Site-directed mutagenesis studiesneighboring subunit, monomerization of the natural tet- also demonstrate the importance of this residue for tight biotinrameric streptavidin can generate streptavidin with re- binding and subunit communications (6 – 8). Therefore, devel-duced biotin binding affinity. Three residues, valine 55, opment of monomeric (strept)avidin can be an attractive ap-threonine 76, and valine 125, were changed to either proach to engineer (strept)avidin with reversible biotinarginine or threonine to create electrostatic repulsion binding capability. Downloaded from www.jbc.org by Juan Slebe on August 14, 2007and steric hindrance at the interfaces. The double mu- The engineering of (strept)avidin to its monomeric form istation (T76R,V125R) was highly effective to monomerize technically challenging. In the case of avidin, the first genera-streptavidin. Because interfacial hydrophobic residues tion of engineered monomeric avidin can exist in the mono-are exposed to solvent once tetrameric streptavidin is meric state only in the absence of biotin (9). This problem hasconverted to the monomeric state, a quadruple mutein(T76R,V125R,V55T,L109T) was developed. The first two been solved by the recent development of the second generationmutations are for monomerization, whereas the last two of monomeric avidin (10), which carries two mutationsmutations aim to improve hydrophilicity at the inter- (N54A,W110K). Structural alignment of avidin and streptavi-face to minimize aggregation. Monomerization was con- din indicates that these two residues correspond to Asp-61 andfirmed by four different approaches including gel filtra- Trp-120 in streptavidin (11). However, a streptavidin muteintion, dynamic light scattering, sensitivity to proteinase designated AK,1 which carries the corresponding double muta-K, and chemical cross-linking. The quadruple mutein tions (D61A,W120K), does not become monomeric as demon-remained in the monomeric state at a concentration strated in the present study. This illustrates the need to iden-greater than 2 mg/ml. Its kinetic parameters for interac- tify a new set of critical residues in combination with effectivetion with biotin suggest excellent reversible biotin bind- approaches to generate monomeric streptavidin with minimaling capability, which enables the mutein to be easily mutational changes. Development of monomeric streptavidinpurified on the biotin-agarose matrix. Another mutein has been reported previously through mutation of three resi-(D61A,W120K) was developed based on two mutations dues to alanine (12). However, the low affinity of this muteinthat have been shown to be effective in monomerizing toward biotin (Kd ϭ 1.7 ϫ 10Ϫ6 M) makes it less than ideal foravidin. This streptavidin mutein was oligomeric in na- many applications.ture. This illustrates the importance in selecting the To develop better versions of monomeric streptavidin, threeappropriate residues and approaches for effective mo- residues (Val-55, Thr-76, and Val-125) were selected for site-nomerization of streptavidin. directed mutagenesis. In combination with L109T mutation, a series of single, double, and quadruple streptavidin muteins were created and produced from Bacillus subtilis via secretion. (Strept)avidin with reversible biotin binding capability can As they were produced in the soluble form without the require-extend the applications of the biotin-(strept)avidin technology. ment of refolding (13, 14), their oligomeric state can be rapidlyThese molecules can be applied for affinity purification of bi- analyzed by SDS-PAGE using culture supernatants from theotinylated biomolecules, screening of ultratight binders bind- streptavidin mutein production strains. The muteins were pu-ing to biotinylated biomolecules displayed on the phage display rified and further characterized by different approaches tosystem, and development of reusable biosensor chips, protein/ confirm their monomeric state. Their kinetic parameters forantibody microarrays, and enzyme bioreactors (1, 2). (Strept)a- biotin binding were determined by surface plasmon resonance-vidin is a homotetrameric molecule with a biotin binding site in based biosensor studies. EXPERIMENTAL PROCEDURES * This work was supported by a discovery grant from the NaturalSciences and Engineering Research Council of Canada and a short term Construction of Streptavidin Mutants—Different point mutationsproject grant from the University of Calgary. The costs of publication of were introduced to the coding sequence of a synthetic streptavidin genethis article were defrayed in part by the payment of page charges. This (ssav) in the B. subtilis expression vector pSSAV-Tcry (14) by PCR-article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. □ The on-line version of this article (available at http://www.jbc.org) S 1 The abbreviations used are: AK, streptavidin mutein with doublecontains Supplemental Figs. S1, S2, and S3. mutations (D61A,W120K); ka, on rate; kd, off rate; Kd, dissocia- ‡ To whom correspondence should be addressed: Dept. of Biological tion constant (kd/ka); M1, streptavidin mutein with a single muta-Sciences, Division of Cellular, Molecular and Microbial Biology, Uni- tion (T76R); M2, streptavidin mutein with double mutationsversity of Calgary, 2500 University Dr., N. W. Calgary, Alberta T2N (T76R,V125R); M4, streptavidin mutein with four mutations1N4, Canada. Tel.: 403-220-5721; Fax: 403-289-9311; E-mail: slwong@ (T76R,V125T,V55T,L109T); PBS, phosphate-buffered saline; sulfo-ucalgary.ca. EGS, ethylene glycol bis(sulfosuccinimidyl succinate).This paper is available on line at http://www.jbc.org 23225
  • 2. 23226 Engineering of Monomeric Streptavidin TABLE I Mutagenic 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 5Ј-GGAAAAGTACTCTTA(C/G)AGGACATGATACATTTAC-3Ј Backward primer pUB18H3 5Ј-GATTTCATACACGGTGCCTG-3Ј V55R,V55T (XbaI/SphI) Forward primer SAVV55RTF 5Ј-GAATCTAGATACA(C/G)ACTTACAGGAAGATATG-3Ј Backward primer pUB18H3 T76R (BamHI/SphI) Forward primer SAVT76RF 5Ј-GTGGATCCGGAACAGCACTTGGATGGAGAGTT-3Ј Backward primer pUB18H3 D61A,W120K (XbaI/ScaI) Forward primer SAVD61AF 5Ј-CATCTAGATACGTGCTTACAGGAAGATATGCATCTGCACCT-3Ј Backward primer SAVW120KB 5Ј-CAAGAGTACTTTTTTTTGCATTTGCTTC-3Ј T76R,L109T (BamHI/ScaI) Forward primer SAVT76RF Backward primer SAVL109TB 5Ј-GAGAGTACTTTTCCATGCATTTGCTTCTGTTGTTCCAGATGTTAATGTCCATTGTGTG-3Ј Downloaded from www.jbc.org by Juan Slebe on August 14, 2007based oligonucleotide-directed mutagenesis. Five mutants (V125R, containing 5 mM CaCl2, pH 8.0. The reaction was stopped by precipita-V125T, V55R, V55T, and T76R), each bearing a single mutation that tion with trichloroacetic acid (18). Boiled samples of precipitated pro-results in the change of an amino acid residue as the name suggests, teins were resolved by reducing SDS-PAGE. The same analysis waswere constructed using pSSAV-Tcry as the template and the primers performed with streptavidin samples treated with biotin (1 mM finallisted in Table I. The amplified products were digested with the pair of concentration) prior to proteinase K digestion.enzymes listed in Table I and cloned into pSSAV-Tcry. Five plasmids Cross-linking Reactions—Cross-linking of streptavidin and its mu-(pV125R, pV125T, pV55R, pV55T, and pT76R) resulted. teins was carried out using ethylene glycol bis(sulfosuccinimidyl succi- Two double mutants (M2 and AK) were also constructed. For M2 nate) (sulfo-EGS) (Pierce) as the cross-linker. A typical reaction mixture(T76R,V125R), a ScaI/NheI-digested fragment of pV125R was used to (20 ␮l) contained the purified mutein (0.25 mg/ml) and sulfo-EGS (10-replace the corresponding fragment in pT76R. For AK (D61A,W120K), fold molar excess over the protein) in PBS. After 30 min at roomthe fragment bearing the two mutations was amplified by PCR using temperature, the reaction was quenched with Tris-HCl (30 mM, pH 7.5).the primers SAVD61AF and SAVW120KB (Table I) and the template Aliquots of the cross-linking reaction samples were boiled and exam-pSSAV-Tcry. The amplified fragment was digested by XbaI/ScaI and ined by SDS-PAGE. Lysozyme (Sigma, 0.25 mg/ml) was included in theused to replace the corresponding fragment in pSSAV-Tcry. study to help establish the optimal reaction conditions. The construction of M4 (T76R,V125R,V55T,L109T) involved two Kinetic Analysis of Streptavidin Muteins—The kinetic parameterssteps (Supplemental Fig. S1). First, a 164-bp fragment bearing two (both on and off rates for interaction with biotin) of streptavidin mu-mutations (T76R,L109T) was amplified using SAVT76RF and teins were determined in real time using the surface plasmon reso-SAVL109TB (Table I) as primers and pSSAV-Tcry as template. The nance-based BIAcoreX biosensor. Biotin-conjugated bovine serum albu-amplified product was digested by BamHI/ScaI and used to replace the min immobilized on a CM5 sensor chip was used to study thecorresponding fragment in pV55T to generate pV55T-T76R-L109T. In reversibility of biotin binding (12).the second step, a ScaI/NheI-digested fragment of pV125R was used to Computer Programs for Streptavidin Analyses—Swiss-pdb Viewerreplace the corresponding fragment in pV55T-T76R-L109T to (19) was used to display streptavidin (Protein Data Bank code 1SWEgenerate pV55T-T76R-L109T-V125R. (20)), analyze interfacial residues, measure distance between residues, Production and Purification of Streptavidin—Wild-type streptavidin and align the structures of streptavidin and avidin. Interfacial contactwas produced by B. subtilis WB800(pSSAV-Tcry) cultured in a defined areas were calculated using the protein-protein interaction server (21)medium (14). The secreted protein was purified to homogeneity using and the Formiga module in the Sting Millennium Suite (22). The plotscation exchange followed by iminobiotin affinity chromatography (12). of accessible surface area of individual residues in streptavidin in eitherProduction and purification of streptavidin muteins followed a similar the monomeric or tetrameric state were generated using the Proteinscheme with two major modifications: super-rich medium (15) was used Dossier module in the Sting Millennium Suite.in place of the defined medium, and biotin-agarose (Sigma) was used inplace of iminobiotin-agarose as the affinity matrix. Dialyzed sample RESULTScontaining partially purified mutein was loaded to a 1-ml biotin-agarose Selection of Key Residues in Streptavidin for Site-directedcolumn. Streptavidin muteins were eluted from the column using 20 Mutagenesis—Tetrameric streptavidin is arranged as a dimermM D-biotin in phosphate-buffered saline (PBS; 50 mM sodium phos- of dimers (Fig. 1A). The interface between subunits A and Bphate, 100 mM NaCl, pH 7.2). Concentration of purified streptavidinwas determined spectrophotometrically using the known extinction (and between C and D) has the most extensive subunit inter-coefficient at 280 nm (16, 17) for each individual mutein. actions. The interfacial contact area between A and B is ϳ1,557 Determination of the Molecular Size of Streptavidin—Molecular Å2 with 17 H-bonding interactions, two salt bridges, and nu-mass of purified streptavidin and its muteins was estimated by both gel merous van der Waals interactions. The interface contact be-filtration and dynamic light scattering studies. Gel filtration was per- tween A and D is also extensive with a contact area of 525 Å2formed on the Bio-Rad biologic work station equipped with a Bio-Prep and two interfacial H-bonding interactions. The weakest inter-SE 100/17 column that had been calibrated with molecular mass pro-tein markers (Bio-Rad). Molecular mass was also estimated from the face interaction is between subunits A and C with an interfa-hydrodynamic radius of the mutein obtained using a DynaPro MS cial contact area of 171 Å2. To engineer monomeric streptavidindynamic light scattering instrument (Protein Solutions) that had been with a minimal number of mutated residues, an attractivecalibrated with lysozyme. Protein samples (2–3 mg/ml in PBS) were approach is to introduce both charge repulsion and steric hin-passed through a 0.02-␮m filter (Whatman Anodisc 13) immediately drance at these interfaces. As protein has structural plasticityprior to measurement. The size distribution profile was analyzed using (23–25), it is vital to select interfacial residues located on athe manufacturer’s Dynamics V6 software. Proteinase K Digestion of Streptavidin and Its Muteins—Purified rigid surface to maximize the effects of charge repulsion andstreptavidin and its muteins (30 ␮M monomer) were treated with pro- steric hindrance. Because streptavidin subunit forms an eight-teinase K (Invitrogen, 5 ␮M) for 15 min at 30 °C in 50 mM Tris-HCl antiparallel stranded ␤-barrel structure (4, 5), the selected
  • 3. Engineering of Monomeric Streptavidin 23227 Downloaded from www.jbc.org by Juan Slebe on August 14, 2007 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.residues should be located on the ␤-strands rather than in the SDS-polyacrylamide gel even in the presence of biotin. In con-loop regions. Furthermore the selected residue in one subunit trast, V55T mutation had the lowest impact with the majorityshould be located very close to the equivalent residue or a of molecules in the tetrameric state even in the absence ofcharged residue in another subunit at the interface. Examina- added biotin. Presence of biotin shifts the majority of the threetion of interfacial residues (Fig. 1, B–D) shows that Thr-76, remaining muteins (V125R, V125T, and V55R) to the tet-Val-125, and Val-55 meet the criteria. Hence they were selected rameric state. As expected, changing valine to arginine exertedfor mutagenesis. greater impact than changing it to threonine. This is true for Effects of Single Mutations on Monomerization of Streptavi- both Val-125 and Val-55.din—Streptavidin muteins carrying a single amino acid change Effects of Multiple Mutations on Monomerization of Strepta-at the selected site were produced in their soluble form by B. vidin—To develop idealized monomeric streptavidin muteinssubtilis via secretion. Analysis of non-boiled culture superna- that are more likely to remain in the monomeric state at hightants by SDS-PAGE offers a quick screen for the mutation streptavidin concentrations and have excellent reversible bio-effect (12). Weaker subunit interaction would result in a higher tin binding capability, two more muteins were created. M2 ispercentage of the sample in the monomeric state on the SDS- the double mutant carrying both the T76R and V125R muta-polyacrylamide gel. Because biotin can strengthen subunit in- tions. M4 is a quadruple mutant carrying T76R, V125R, V55T,teraction, samples were analyzed in the presence or absence of and L109T mutations. In this combination, the three interfa-biotin (9, 26). The impact of the mutation on weakening of the cial hydrophobic residues Val-125, Val-55, and Leu-109 weresubunit interaction followed the order: T76R Ͼ V125R Ͼ changed to hydrophilic ones. The last construct is AK, a doubleV125T Ϸ V55R Ͼ V55T (Fig. 2 and Table II). The T76R mutein mutant (D61A,W120K) carrying two mutations (equivalent to(designated M1) existed 100% in the monomeric state on the those performed in avidin) that have been shown to convert
  • 4. 23228 Engineering of Monomeric Streptavidin Determination of Apparent Molecular Mass of Streptavidin Muteins by Dynamic Light Scattering—Because the apparent molecular mass of wild-type streptavidin in the absence of biotin is 10 kDa less than expected (56 instead of 66 kDa) as determined by gel filtration, dynamic light scattering (29) was used as a second method to estimate the apparent molecular masses. The apparent molecular mass of wild-type streptavidin obtained in this way (69 kDa) was closer to that expected (66 kDa) (Table III). The apparent molecular masses for both M2 and M4 in the absence of biotin indicated that they were in the monomeric state. Addition of biotin caused only a slight in- crease in their apparent molecular masses. The AK mutein again was found to be oligomeric independent of the presence or absence of biotin. Proteinase K Sensitivity of Streptavidin Muteins—Mono- FIG. 2. Western blot analysis of culture supernatants from meric streptavidin is expected to be more susceptible to pro-B. subtilis strains producing streptavidin muteins carrying a teinase K digestion (10). Therefore, wild-type streptavidin andsingle mutation. 15 ␮l of non-boiled sample of culture supernatant its muteins were treated with proteinase K (Fig. 5A). Wild-typewas loaded to each lane. Samples in the left set were collected fromB. subtilis strains cultured in super-rich medium without added biotin. streptavidin was converted to the core form independent of theSamples in the right set were from culture grown in the presence of presence or absence of biotin. Under the condition used, thebiotin (20 ␮M). The blot was probed with polyclonal antibodies against core streptavidin was resistant to further degradation by pro- Downloaded from www.jbc.org by Juan Slebe on August 14, 2007streptavidin. M, molecular weight markers; wt, wild-type streptavidin; teinase K. In contrast, all three muteins including AK, M2, andϪve control, culture supernatant from WB800(pWB705HM) (34) thatdid not produce any streptavidin. M4 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 ortetrameric avidin to the monomeric state (10). As shown in Fig. absence of biotin. This property is consistent with the mono-3 and Table II, just like M1, all these muteins existed in meric nature of these muteins. The AK mutein behaved differ-monomeric state on the SDS-polyacrylamide gel even in the ently. Although most of it was digested by proteinase K in thepresence of biotin. absence of biotin, it became much more resistant to proteinase Purification of Streptavidin Muteins—Purification of M4 was K when biotin was present.used as an example to illustrate the process (Fig. 4). Proteins Cross-linking of Streptavidin and Its Muteins—Topartially purified by ion exchange chromatography (lane 2) strengthen the idea that both M2 and M4 are monomericwere applied to a biotin-agarose column. M4 could be readily whereas AK is oligomeric in nature, protein cross-linking waseluted off from the column using biotin-containing buffer as the carried out using sulfo-EGS as the cross-linking agent. Sulfo-eluant (lanes 5–7). Pure streptavidin mutein obtained by this EGS reacts with both the accessible ␣-amino groups at the Nsimple procedure, after removal of biotin by dialysis, could be termini and the surface-exposed ⑀-amino groups of the lysineused for biochemical characterizations. To demonstrate thatdialysis could effectively remove any bound biotin from the M4 side chains in proteins. Secreted wild-type streptavidin hasmutein, the dialyzed sample was reloaded to the biotin-agarose eight lysine residues in each subunit. The three-dimensionalmatrix. Over 95% of the sample could be retained on the col- structural model of streptavidin suggests that lysine 121 inumn and eluted off from the column using biotin (data not subunit A is 14.1 Å from lysine 121 in subunit D. As the spacershown). Of all the muteins, M1 tended to have a long trailing arm in sulfo-EGS is 16.1 Å, subunits A and D (same for sub-tail during elution. This indicates that M1 may not have the units B and C) should be easily cross-linked by sulfo-EGS. Alsodesirable reversible biotin binding property. Therefore, it was it is possible to have cross-linking between subunits A and B asnot characterized further. the N-terminal region from subunit A, which contains two Determination of Apparent Molecular Mass of Streptavidin lysine residues, is likely to be positioned close to lysine 80 inMuteins by Gel Filtration—Observation of 100% monomeriza- subunit B. The same is true for subunits C and D. Therefore,tion of the streptavidin mutein using a non-boiled sample for one should be able to differentiate tetrameric streptavidin fromSDS-PAGE does not always truly reflect its existence in the the monomeric form with the observation of cross-linked tet-monomeric state in solution because SDS can promote subunit rameric streptavidin using sulfo-EGS. Lysozyme, well knowndissociation (27, 28). The apparent molecular masses of the to be monomeric in solution (30, 31), served as the negativepurified wild-type streptavidin and the three muteins (M2, M4, control. Fig. 5B shows that the amount of dimeric lysozymeand AK) were estimated by gel filtration (Supplemental Fig. 2A increased slightly in the presence of the cross-linking agent.and Table III). The expected molecular mass of monomeric This helped set the upper limit of the concentration of sulfo-streptavidin is 16.5 kDa. M2 and M4 in the absence of biotin EGS to be used under the experimental condition. The wild-showed the apparent molecular masses of 19.95 and 21.87 kDa, type streptavidin subunit had an apparent molecular mass ofrespectively. These masses increased slightly in the presence of 19 kDa on the SDS gel. After treatment with sulfo-EGS, mostbiotin. These data suggest that the muteins are monomeric in of these subunits were cross-linked to dimers and higher oli-nature because their masses are less than that for the strepta- gomers with small amounts remaining in the monomeric state.vidin dimer (33 kDa). In contrast, the AK mutein showed an M2 and M4 muteins behaved very similarly (data for M2 areapparent molecular mass of 45.66 kDa even in the absence of not shown). The majority of the M2 and M4 muteins after thebiotin. This indicates the oligomeric nature of this mutein. cross-linking treatment migrated as monomers with smallSupplemental Fig. 2B shows the elution profile of purified M4 amounts in the dimeric form. These dimers may represent(in the absence of biotin) from the gel filtration column. The cross-linked monomeric subunits that were artificially gener-sample (loaded at 2 mg/ml) was eluted as a single peak. There ated in the same manner as with lysozyme. AK showed ais no evidence for the presence of tetrameric streptavidin, cross-linking profile very similar to that of the wild-typewhich would be eluted at 30.5 min. streptavidin. These data strongly support the idea that M2 and
  • 5. Engineering of Monomeric Streptavidin 23229 TABLE II Summary of the mutagenic effects on weakening of subunit interactions in streptavidin muteins as reflected by the degree of monomerization of streptavidin muteins after SDS-PAGE Estimation of the percentage of streptavidin in monomeric and tetrameric states is based on the blots showing the migration pattern ofnon-boiled samples in Figs. 2 and 3. No additional biotin With additional biotin Streptavidin mutein Monomer Tetramer Monomer Tetramer % M1 (T76R) 100 0 100 0 V125R 92 8 12 88 V125T 25 75 0 100 V55R 20 80 10 90 V55T 1 99 0 100 M2 (T76R,V125R) 100 0 100 0 M4 (T76R,V125R,V55T,L109T) 100 0 100 0 AK 100 0 100 0 TABLE III Molecular mass determination of wild-type (wt) streptavidin and its muteins by gel filtration and dynamic light scattering In 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%. Downloaded from www.jbc.org by Juan Slebe on August 14, 2007 Theoretical molecular mass is estimated from the amino acid composi- tion of the mature protein (17). Gel Dynamic light Sample filtration scattering Theoretical M M RH M kDa nm kDa kDa wt (no biotin) 56.23 3.69 Ϯ 0.19 69 66.0 (tetramer) FIG. 3. Analysis of culture supernatants from B. subtilis AK (no biotin) 45.66 3.20 Ϯ 0.23 50strains producing streptavidin muteins carrying different com- AK (ϩbiotin) 50.12 3.54 Ϯ 0.38 63binations of mutations. All these strains were cultivated in super- M2 (no biotin) 19.95 1.98 Ϯ 0.24 17 16.5 (monomer)rich medium supplemented with biotin (20 ␮M). 15 ␮l of non-boiled M2 (ϩbiotin) 23.44 2.09 Ϯ 0.29 18sample of culture supernatant was loaded. A, Coomassie Blue-stained M4 (no biotin) 21.87 2.08 Ϯ 0.31 18.1 16.5 (monomer)SDS-polyacrylamide gel. Bands corresponding to tetrameric and mono- M4 (ϩbiotin) 24.54 2.20 Ϯ 0.14 21.5meric 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, DISCUSSIONnegative control. Although streptavidin and avidin have similar three-dimen- sional structures and biotin binding properties, development of monomeric 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 buried at the interface in tetrameric streptavidin. This can potentially affect the solubility of the monomeric streptavidin and lead to reassociation of the monomers. The problem can be less dra- FIG. 4. Purification of the M4 streptavidin mutein using bio- matic for avidin, which is a glycosylated protein with a carbo-tin-agarose. M4 mutein partially purified by Macro S column chroma- hydrate chain in each of the avidin subunits.tography (PPF) was loaded on a biotin-agarose column. M, molecular Despite the challenge, our study illustrates that, by selectingmass markers; FT, column flow-through; W, pooled washing fractions; a critical residue located on a rigid surface for mutagenic studyEF1–EF3, eluted fractions. and the introduction of charge repulsion and steric hindrance at the interface, a single mutation (T76R) can be greatly effec-M4 muteins are monomeric, whereas the AK mutein is oligo- tive in developing monomeric streptavidin. Besides the sugges-meric in solution. tion from SDS-PAGE analysis, gel filtration study of the M1 Reversible Interaction between Streptavidin Muteins and mutein also indicated that the majority of M1 was eluted at aBiotin—The on rate and off rate of the interactions between position corresponding to the monomeric form (data notstreptavidin muteins and biotin were determined by surface plas- shown). The main drawback for this mutein is its elution be-mon resonance-based BIAcore biosensor (12). As shown in Table havior on the biotin-agarose column. The elution profile had aIV (graphical plots for M4 are shown in Supplemental Fig. S3), typical long trailing tail. Furthermore more M1 could be recov-M2, M4, and AK had their dissociation constant (Kd) in the range ered by soaking the column overnight with buffer containingof 10Ϫ7 M. The off rates (kd) for these muteins were almost the biotin. This suggests that some of the M1 population havesame, whereas the on rate (ka) for the AK mutein was slightly higher affinity to the matrix.lower than the rest. One of the factors affecting the on rate is the To increase the efficiency of streptavidin monomerization,diffusion coefficient (or molecular mass) of the streptavidin mol- M2 mutein was developed by combining two potent mutationsecule. Because AK is oligomeric in nature, this may account for (T76R,V125R). Data from gel filtration study, dynamic lightthe lower on rate for this mutein-biotin interaction. scattering, sensitivity to proteinase K, and cross-linking reac-
  • 6. 23230 Engineering of Monomeric Streptavidin 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. TABLE IV gation of the mutein. Another attractive feature is that M4 has Kinetic parameters for the interactions between streptavidin a remarkably sharp elution profile with its purification using muteins and biotin biotin-agarose. Over 95% of the mutein could be readily elutedProtein ka kd Kd off from the column using just 2 column volumes of the eluant, Ϫ1 leading to a high rate of protein recovery. M sϪ1 sϪ1 M Downloaded from www.jbc.org by Juan Slebe on August 14, 2007 In the site-directed mutagenesis study, it is not difficult to M2 1.88 Ϯ 0.07 ϫ 104 3.22 Ϯ 0.01 ϫ 10Ϫ3 1.71 Ϯ 0.07 ϫ 10Ϫ7 M4 1.80 Ϯ 0.04 ϫ 104 3.39 Ϯ 0.02 ϫ 10Ϫ3 1.87 Ϯ 0.06 ϫ 10Ϫ7 understand the impact of the mutations in the following order: AK 1.46 Ϯ 0.05 ϫ 104 3.59 Ϯ 0.03 ϫ 10Ϫ3 2.46 Ϯ 0.10 ϫ 10Ϫ7 T76R Ͼ V125R Ͼ V55R. Analysis of the solvent accessibility of individual amino acid residues with tetrameric streptavidin indicates that the solvent-accessible area of Thr-76 in subunittion confirmed the monomeric state of M2. The elution profile A is zero. Its close distances to both Thr-76 and Arg-59 inof this mutein from the biotin-agarose matrix had a consider- subunit B and location on a rigid surface of a ␤-barrel structureable improvement over that of M1. When a 30-min on-column make it an ideal residue to be changed to arginine to achieveincubation period was allowed for the eluant in between frac- the maximal electrostatic repulsion and steric hindrance ef-tion collection, about 90% of M2 could be recovered within 3 fects at the subunit interface (Fig. 1B). The surface-accessiblecolumn volumes of the eluant. area of Val-125 in subunit A is 1.78%. It has extensive inter- To ensure that the streptavidin mutein will stably remain in actions with Leu-109, Trp-120, Thr-123, and Val-125 in sub-the monomeric state, more mutations were introduced to M2. unit D (Fig. 1C); Leu-109 in subunit B; and Gln-107 in sub-The exposure of the AB (or CD) interface will expose three unit C. Its conversion to arginine results in charge repulsionhydrophobic residues: Val-55, Leu-109, and Val-125. In the M2 in subunit D and potential steric hindrance for subunits B, C,mutein, Val-125 has been changed to arginine. We chose to and D. As for Val-55 in subunit A, its surface accessible areafurther convert Val-55 and Leu-109 to threonine, a more hy- is 29.7%; and it is only close to Arg-59 in subunit B at thedrophilic residue, to develop the M4 mutein. Conversion to interface (Fig. 1D). Thus, V55R has the least impact onthreonine instead of arginine is preferred because proteins monomerization.with high pI are known to have nonspecific interactions via Although AK mutein shows reversible biotin binding prop-electrostatic interactions (32, 33). The calculated pI of M2 is erty and monomeric behavior on the SDS-polyacrylamide gel, it8.1. If both Val-55 and Leu-109 are converted to arginine, the clearly exists in the oligomeric state in solution as suggested byresulting mutein will have a pI of 9.2. This may increase the gel filtration studies, dynamic light scattering, and cross-link-chance of charge-related nonspecific interactions. The conver- ing pattern. This study illustrates the importance of selectingsion of these residues to negatively charged residues was not critical residues and effective approaches to achieve the maxi-considered because both Val-55 and Leu-109 are located on the mal monomerization effect on tetrameric streptavidin.␤-strands, and negatively charged residues are relatively poor␤-strand formers. REFERENCES M4 mutein shares with M2 mutein many desirable features 1. Morag, E., Bayer, E. A., and Wilchek, M. (1996) Anal. Biochem. 243, 257–263 2. Balass, M., Morag, E., Bayer, E. A., Fuchs, S., Wilchek, M., and Katchal-of an idealized monomeric streptavidin. They both exist in the skikatzir, E. (1996) Anal. Biochem. 243, 264 –269monomeric state at a reasonably high protein concentration (2 3. Green, N. M. (1990) Methods Enzymol. 184, 51– 67mg/ml or more as used in the dynamic light scattering study). 4. Weber, P. C., Ohlendorf, D. H., Wendoloski, J. J., and Salemme, F. R. 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