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Journal of Biotechnology
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amavidin 2-REV: An engineered tamavidin with reversible biotin-bindingapability�
oshimitsu Takakura ∗, Kozue Sofuku, Masako Tsunashimalant Innovation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan
r t i c l e i n f o
rticle history:eceived 21 August 2012eceived in revised form8 December 2012ccepted 9 January 2013vailable online 17 January 2013
eywords:ffinity purification
a b s t r a c t
A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinitypurification of biotinylated biomolecules. Although several proteins, chemically or genetically modifiedfrom avidin or streptavidin, with reversible biotin-binding have been reported, they have been prob-lematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin inbiotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the ser-ine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound tobiotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate
biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coliby means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotinwas 2.8–4.4 × 10−7 M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, suchas high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating
pow
that tamavidin 2-REV is a
. Introduction
Avidin, from chicken, and its bacterial analogue, streptavidin,rom Streptomyces avidinii are homotetrameric proteins that bindiotin. Each subunit binds one molecule of biotin, and, with a Kd of
× 10−16 M for avidin and a Kd of 4 × 10−14M for streptavidin, theseffinities are the highest known in nature for a ligand and a proteinGreen, 1990). The extraordinarily high affinity and specificity ofhe (strept)avidin–biotin interaction is of great technical value andas been exploited in diverse applications in medicine, biology, bio-hemistry, and biotechnology (Schetters, 1999; Wilchek and Bayer,990). However, in certain applications, such as affinity chromatog-aphy for the purification of biotinylated biomolecules, this highffinity is a drawback. The binding of (strept)avidin and biotin is vir-ually irreversible under physiological conditions, and only harsh
reatments, which may inactivate many biomolecules, induce dis-ociation. Therefore, a biotin-binding protein that reversibly bindsiotin has been eagerly sought.
� Note: nucleotide sequence data are available in the DDBJ/EMBL/GenBankatabases under the accession number AB738087 for tam2-S36A.∗ Corresponding author. Present address: Leaf Tobacco Research Center, Japan
Numerous proteins with a lower affinity to biotin have beenproduced by engineering avidin or streptavidin chemically (Moraget al., 1996) or genetically (reviewed in Laitinen et al., 2006). Inthese proteins, the residues that associate with biotin throughhydrogen bonding (Hyre et al., 2000; Kopetzki et al., 1997; Moraget al., 1996; Qureshi et al., 2001; Qureshi and Wong, 2002) orhydrophobic interactions (Chilkoti et al., 1995; Laitinen et al., 1999;Sano and Cantor, 1995) were modified. In addition, because avidinand streptavidin exhibit strong biotin binding only in the tetramericform, the residues involved in the inter-subunit contacts havealso been modified (Laitinen et al., 2003; Wu and Wong, 2005).Some of these engineered proteins bound biotin reversibly, and afew actually released biotinylated biomolecules under mild condi-tions (Malmstadt et al., 2003; Qureshi and Wong, 2002; Wu andWong, 2006). However, these engineered proteins all had short-comings, such as they were produced inefficiently, were sensitiveto proteases, prone to form aggregates, or they had insufficientreversibility or high isoelectric points that could increase thechance of charge-derived non-specific binding to other molecules(Marttila et al., 2000).
One of these engineered proteins, streptavidin-S45A, in whichthe serine residue at position 45 (S45) of streptavidin was replaced
with an alanine residue, has been studied extensively (Hyre et al.,2000, 2006). In the presence of excess free biotin, the T1/2 (thetime in which half of the protein dissociated tritiated biotin)of streptavidin-S45A was 1.3 min, whereas that of streptavidin
Fig. 1. Interaction of S36 with biotin and V38 in the tamavidin 2-biotin complex.Rwy
wsoeacp
masEntvbvbptipa
ma
2
2
t5sa3fTsa3t
esidue S36 forms hydrogen bonds with main chain V38 (NH and C O) as well asith the ureido-nitrogen of biotin. Predicted hydrogen bonds are represented by
ellow broken lines.
as 3001 min (Hyre et al., 2006). Moreover, the application oftreptavidin-S45A in affinity thermo-precipitation and recoveryf biotinylated biomolecules has been demonstrated (Malmstadtt al., 2003). These findings were remarkable in that a singlemino acid substitution caused a drastic change in the asso-iation/dissociation kinetics, inspiring similar studies of otherroteins.
Tamavidin 2, another avidin-like tetramer, is found in edibleushroom (Pleurotus cornucopiae) and binds biotin as strongly as
vidin and streptavidin (Takakura et al., 2009). Unlike avidin andtreptavidin, it can be expressed in soluble form at high levels inscherichia coli (Takakura et al., 2009) and therefore may be engi-eered and produced efficiently. Crystallographic analysis of theamavidin 2/biotin complex revealed that seven residues of tama-idin 2 (N14, S18, Y34, S36, S76, T78, and D116) form hydrogenonds with biotin and that S36 is the counter part of S45 in strepta-idin. S36 forms a hydrogen bond with the ureido-nitrogen of theiotin bicyclic ring. It also forms hydrogen bonds with the valine atosition 38 (V38) (Fig. 1), which is located in the loop region (S36o V41) between the third and fourth �-strands of tamavidin 2. Its possible that this loop functions as a lid for the biotin bindingocket and is reinforced by the two hydrogen bonds between S36nd V38.
Here, we report that a mutein of tamavidin 2 bearing the S36Autation, designated as tamavidin 2-REV, reversibly bound biotin
nd retained the other useful characteristics of tamavidin 2.
. Materials and methods
.1. Construction of the gene for tamavidin 2-REV
The 5′-portion of the mutant gene was amplified from theamavidin 2 cDNA (Takakura et al., 2009) between the primer Tm2-′Pci (5′-AAAACATGTCAGACGTTCAATCTTC-3′) for the 5′-terminalequence of the open reading frame and a restriction site (PciI)nd the antisense primer (5′-ATCCCCAACTTTCGCGAGGTACTTTCC-′) for the mutated sequence. The 3′-portion was amplifiedrom the same template between the primer Tm2-3′Bam (5′-TTTTTGGATCCTTACTTCAACCTCGGTGCG-3′) for the 3′-terminal
equence of the open reading frame and a BamHI restriction sitend the sense primer (5′-GGAAAGTACCTCGCGAAAGTTGGGGAT-′), which was complementary to the antisense primer. A mixture ofhe PCR products purified via agarose gel electrophoresis served as
chnology 164 (2013) 19– 25
templates for the second PCR between Tm2-5′Pci and Tm2-3′Bam.The resultant PCR product was digested with PciI and BamHI andcloned into the expression vector pTrc99A (Amersham Biosciences,Piscataway, NJ) predigested with NcoI and BamHI. The plasmid wasintroduced into E. coli strain BL21, and the sequence was verified.
2.2. Expression in E. coli
Tamavidin 2-REV was expressed in E. coli according to the pro-cedure described by Takakura et al. (2009). The E. coli strain wascultured in Luria-Bertani broth containing 1 mM IPTG at 25 ◦Covernight with vigorous shaking.
2.3. Affinity chromatography
Total soluble protein was extracted from 25 mL of the E. coli cul-ture by ultrasonic homogenization in 1.5 mL of 100 mM potassiumphosphate buffer (pH 7.0). The cell debris was removed by centrifu-gation at 15,000 × g. Then, 400 �L of biotin agarose (Sigma, St. Louis,MO) was added to the supernatant, and the container was contin-uously inverted for 1 h. The biotin agarose was packed in a column(0.5 cm in diameter × 3 cm in length), washed with 10 mL of PBS (pH7.4) that contained 500 mM NaCl and then eluted three times with0.5 mL of PBS (pH 7.4) that contained 10 mM biotin. Iminobiotincolumn chromatography was performed according to the proce-dure described by Takakura et al. (2009). The eluted fractions weredialyzed overnight against 20 mM potassium phosphate buffer (pH7.0). The proteins were separated by 15% SDS-PAGE and stainedwith Coomassie® brilliant blue (CBB) G-250 (Sigma). The amount ofprotein was estimated by densitometry. The recovery rate (%) wasdefined as the ratio of the amount of mutein after/before purifica-tion, and purity (%) was defined as the ratio of the amount of muteinto total soluble protein in the purified fraction.
2.4. Gel filtration
Tamavidin 2-REV was applied on a Sephacryl S-100HR (GEHealthcare, Piscataway, NJ) with molecular weight markers [GelFiltration Calibration Kit LMW consisting of Aprotinin (Mr 6500),Ribonuclease A (Mr 13700), Carbonic anhydrase (Mr 29000), Oval-bumin (Mr 43000), Conalbumin (Mr 75000), and Blue dextran2000 (Mr 2000000); GE Healthcare] in 50 mM potassium phosphatebuffer (pH 7.0) containing 500 mM NaCl.
2.5. Protease assay
The protease assay was performed as described by Wu andWong (2005). Protein (10 �M) was incubated at 30 ◦C for 15 minwith protease K (1.6 �M) in 50 mM Tris–HCl (pH 8.0) that contained5 mM CaCl2 with or without 1 mM biotin. Then, 2× SDS samplebuffer was added to each reaction, and the reaction was terminatedby heat treatment at 95 ◦C for 10 min. The sample was subjected toSDS-PAGE and stained with CBB.
2.6. Thermal stability
The protein (1.0 mg/mL in 20 mM potassium phosphate buffer,pH6.5) was treated at 25 ◦C, 65 ◦C, 70 ◦C, 75 ◦C, 80 ◦C, 85 ◦C, 90 ◦C,and 99.9 ◦C for 20 min, and then centrifuged at 10,000 × g for 10 minto pellet the denatured protein. The protein in the supernatant was
mixed with the same amount of 2× SDS sample buffer, treated at95 ◦C for 20 min, subjected to SDS-PAGE, and stained by CBB. Theband corresponding to the monomer was analyzed by using LAS-3000 imaging analyzer (FujiFilm, Japan). Tm, the temperature at
f Biotechnology 164 (2013) 19– 25 21
wo
2
Tcmuw3tTHHs6ratubsctb(db
2
iwwtiicotcTsr1tb
2
iaw1arw7gpfc
Fig. 2. Purification of tamavidin 2-REV by using biotin-agarose. Tamavidin 2 (A) ortamavidin 2-REV (B) was applied to a biotin-agarose column. After washing, the
Y. Takakura et al. / Journal o
hich the amount of the protein in the heated sample became halff that in the non-heated (at 25 ◦C) sample, was calculated.
.7. Analysis of protein–biotin interactions and kinetics
Kinetic parameters (ka and kd) were evaluated with a Biacore®
100 biosensor based on surface plasmon resonance (GE Health-are UK Ltd., Amersham Place, England) by a single-cycle kineticsethod. Tamavidin 2-REV was immobilized to 5258 resonance
nits on a CM5 sensor chip using the amine coupling method. Biotinas used as the sample at concentration of 10 nM, 30 nM, 100 nM,
00 nM, and 1000 nM. Reagents other than the protein were addedo a reference cell as a negative control for background correction.he biotin was passed through flow cells in a running buffer [10 mMEPES pH 7.4, 150 mM NaCl, 3 mM EDTA 0.005% Surfactant 20 (GEealthcare UK Ltd.)] at a flow rate of 100 �l/min for 2 min. Dis-
ociation of the sample was monitored in the running buffer for0 min. All measurements were performed at 25 ◦C. Associationate constants (ka) and dissociation rate constants (kd) for the inter-ctions between tamavidin 2-REV and biotin were calculated fromhe sensorgrams obtained by a non-linear, global fitting methodsing the Biacore® T100 Evaluation software, version 2.0.1. The 1:1inding model was adopted. The sensorgram was corrected by theubtraction of sensorgram of a reference cell from that of a ligandell. Furthermore, the sensorgram was corrected by the subtrac-ion of sensorgram of the blank sample (running buffer) measuredefore the injection of biotin samples. The dissociation constantKd) was calculated from kd/ka or from the equilibrium-responseata. A molecular mass of 244.3 was used in the calculation foriotin.
.8. Immobilization of tamavidin 2-REV to SepharoseTM
SepharoseTM resin (1 mL) activated with N-hydroxy succin-mide (NHS) (HiTrapNHS-activated HP, GE Healthcare) was treated
ith isopropanol and then 1 mM HCl for activation and was thenashed with distilled water, according to manufacturer’s instruc-
ions. Then, 1.17 mg of tamavidin 2-REV, which was purified byminobiotin column chromatography, was added to the resin andncubated at room temperature for 3 h with gentle shaking. Afterentrifugation at 2000 × g, the supernatant was removed, and 5 mLf 50 mM Tris–HCl (pH 8.0) was added to the resin, which washen further shaken at room temperature for 2 h. The resin wasollected by centrifugation at 2000 × g, and 5 mL of 0.5% BSA/0.05%ween 20 was added to it as a blocking agent. It was then furtherhaken for 30 min. After being washed with 5 mL of PBS (pH 7.4), theesin was resuspended in 1 mL of PBS (pH 7.4). The resin retained
mg (calculated from 1.17 mg minus the quantity of unbound pro-ein) of tamavidin 2-REV, which theoretically contains 70 nmol ofiotin-binding sites. Thus, the coupling efficiency was 86%.
.9. Purification of biotinylated BSA
E. coli TB1 cells from a 25-mL overnight culture were suspendedn 1.5 mL of 100 mM sodium phosphate buffer (pH 7.0), sonicated,nd centrifuged at 15,000 × g. Then, 300 �L of the supernatant,hich contained ca. 3 mg of total soluble proteins, was mixed with
.66 �g of the biotinylated BSA, which consisted of 25 pmol of BSAnd ca. 250 pmol of biotin. The mixture was applied to 75 �L of theesin bearing tamavidin 2-REV immobilized to SepharoseTM, whichas pre-equilibrated with 100 mM sodium phosphate buffer (pH
.0). The resin was gently shaken for 1.5 h, collected by centrifu-
ation at 2000 × g for 15 s, and washed with 1 mL of 0.1 M sodiumhosphate buffer (pH 7.0) three times. The resin was then incubatedor 15 min in 150 �L of 0.1 M sodium phosphate buffer (pH 7.0) thatontained 5 mM biotin to elute the biotinylated BSA. This elution
protein was eluted by adding 10 mM biotin. Fractions were subjected to SDS-PAGEand then stained with Coomassie brilliant blue (CBB). T, total soluble fraction beforebinding the resin; FT, column flow-through; W, wash; E, eluate.
step was repeated once. The same experiment was performed with1.66 �g of biotinylated BSA in 300 �L of 100 mM sodium phosphatebuffer (pH 7.0) alone and 75 �L of resin. In this case, the elution wasperformed only once with 300 �L of the buffer containing biotin.Sample aliquots prior to application, supernatants after centrifu-gation, washes, and eluates were all subjected to SDS-PAGE andproteins were visualized by using a Silver Staining kit II (Wako PureChemicals, Osaka, Japan). The recovery rate (%) was calculated fromthe amount of BSA before and after purification. The purity (%) wasdefined as the ratio of the amount of BSA to the total amount ofprotein in the purified fraction.
2.10. General methods
DNA was manipulated according to the methods describedby Sambrook and Russell (2001). Restriction endonucleases wereobtained from Takara Biochemicals (Shiga, Japan) or Roche Diag-nostics K.K. (Tokyo, Japan). DNA and amino acid sequences wereanalyzed by using GENETYX-WIN ver.8 software (Genetyx Co.,Tokyo, Japan).
3. Results and discussion
3.1. Production and purification of tamavidin 2-REV
The gene for tamavidin 2 was modified by site-directed muta-genesis so that residue S36 was replaced with alanine therebyproducing the mutein tamavidin 2-REV bearing the mutation S36A.The hydrogen bond between S36 and biotin and the two hydrogenbonds between S36 and V38 were expected to be broken by thismutagenesis.
Tamavidin 2-REV was highly expressed as a soluble protein in E.coli. The expression level of between 1 mg and 2 mg from a 50-mLculture was similar to that for tamavidin 2 (Takakura et al., 2009).Affinity column chromatography with 2-iminobiotin-agarose and
biotin-agarose was attempted with the proteins produced in E. coli.Both tamavidin 2 and tamavidin 2-REV were efficiently purifiedwith the 2-iminobiotin-agarose (data not shown), whereas onlytamavidin 2-REV was eluted from the biotin-agarose (Fig. 2). Once
22 Y. Takakura et al. / Journal of Biotechnology 164 (2013) 19– 25
-505
101520253035
-100 -50 0 50 100 150 200 250 300
RUR
espo
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(0 =
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10301003001000nM
05
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0 0. 2 0. 4 0. 6 0. 8 1
RU
Equi
libriu
m R
espo
nse
Biotin concentrati on ( M)µ
Rmax, 43.2 RU
(A)
(B)
Fig. 3. Kinetics of the interaction between tamavidin 2-REV and biotin. Vari-ous concentrations of biotin were added to the tamavidin 2-REV chip, and theinteraction was measured by a Biacore® T100 biosensor. (A) Reversibility of thebvR
ttabcwdv2ec
3b
ekvwwddpBo2tftvw
Fig. 4. Tetrameric structure of tamavidin 2-REV.The apparent molecular mass of tamavidin 2-REV was estimated by gel filtrationchromatography (A) and SDS-PAGE without heat-treatment before sample-loading(B). Arrowheads indicate the peak (A) and the band (B) of tamavidin 2-REV, respec-tively. In the panel (A), figures and arrows at the upper region are the molecularmasses (kDa) of the marker proteins determined separately under the same chro-matographic conditions.
Fig. 5. Protease tolerance of tamavidin 2-REV. The protein, with or without biotin,was reacted with proteinase K at 30 ◦C for 15 min and then subjected to SDS-PAGE.Tamavidin 2 and BSA served as positive and negative controls, respectively.
Fig. 6. Purification of biotinylated BSA by use of tamavidin 2-REV sepharose. A mix-
iotin–tamavidin 2-REV interaction. (B) The equilibrium response (Req) is plottedersus biotin concentration. The Kd of the biotin is equal to the concentration atmax/2.
he extracts were loaded onto the biotin-agarose column, neitheramavidin 2 nor tamavidin 2-REV was detected in the flow-throughnd wash, indicating that both proteins strongly adsorbed to theiotin-agarose. Almost all of the tamavidin 2-REV was eluted effi-iently after the addition of an excess amount of free biotin (Fig. 2B),hereas tamavidin-2 was never eluted (Fig. 2A). This result clearlyemonstrates that unlike tamavidin 2 binding, the binding of tama-idin 2-REV to biotin is reversible under mild conditions. Of note,-iminobiotin-agarose and biotin-agarose yielded similar recov-ry rates (95%) and purity (95%) for tamavidin 2-REV after columnhromatography.
.2. Kinetic parameters for the binding of tamavidin 2-REV toiotin
The interaction between tamavidin 2-REV and biotin wasxamined using the Biacore® T100 biosensor by a Single-cycleinetics method. As shown in Fig. 3A, the binding of tama-idin 2-REV to biotin was completely reversible. The on-rate (ka)as 5.52 × 105M−1 s−1, and the off-rate (kd) was 1.56 × 10−1 s−1,ith a resulting dissociation constant (Kd) of 2.8 × 10−7M. Theissociation constant calculated from the equilibrium-responseata was 4.4 ± 0.8 × 10−7M(Fig. 3B). Compared with the kineticarameters for the binding of tamavidin 2 to biotinylatedSA (ka, 1.0 ± 0.3 × 106M−1 s−1; kd, below the detection limitf 5 × 10−6 s−1) we have ever determined (Takakura et al.,009), the on-rate of tamavidin 2-REV was comparable tohat of tamavidin 2, whereas the off-rate was at least 30,000-
old greater than that of tamavidin 2. Thus, we confirmedhat tamavidin 2-REV has greatly lower affinity than tama-idin 2 for biotin. The obtained Kd values of tamavidin 2-REVere similar to those of monoavidin (1.1–2.9 × 10−7M) and
ture of biotinylated BSA and E. coli total soluble proteins (A) or biotinylated BSA only(B) was applied to tamavidin 2-REV sepharose, washed, and then eluted by adding5 mM biotin. Fractions were subjected to SDS-PAGE and stained with silver. T, totalprotein fraction before application; U, unbound fraction; W, wash; E, eluate.
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et al.
/ Journal
of Biotechnology
164 (2013) 19– 2523
Table 1Modified proteins with reversible biotin-binding capabilities.
Protein Modification Molecularinteractionaffected
Structure Kda (M) Elution of
biotinylatedprotein
Proteaseresistance
pIb Aggregation Yield in E. colic
(mg/L)Comment Reference
Nitro-avidin Nitration ofY33
Hydrogenbonding
Tetramer <10−9 pH 10 NRd NR NR – Incompletechemicalconversion
Morag et al.(1996)
Avidin-W110K W110K Hydrophobicinteraction
Dimer 2.7 × 10−8 NR No 10.3 NR NR Expression ininsect cells
Laitinen et al.(1999)
Monoavidin N54A, W110K Subunitassociation
Monomer 1.1–2.9 × 10−7 NR No 10.1 NR NR Expression ininsect cells
Laitinen et al.(2003)
Streptavidin-W120F W120F Hydrophobicinteraction
Tetramer 0.3–1 × 10−8 NR NR 6.1 NR NR Insoluble inE. coli
Sano andCantor (1995)
Streptavidin-S45A S45A Hydrogenbonding
Tetramer 7.3 × 10−11 Excess biotin NR 6.1 NR NR Insoluble inE. coli
Hyre et al.(2000, 2006)Malmstadt etal. (2003)
Streptavidin-8-aa-loop-H127C 8-aa-loopH127C
Loop7–8,subunitassociation
Tetramer 1.9 × 10−8 Excess biotin NR 4.8 NR NR Expression inBacillus subtilis
O’Sullivan et al.(2012)
Streptavidin-W120K W120K Hydrophobicinteraction
Dimer 6.0 × 10−8 NR Yes 7.6 NR NR Expression ininsect cells
a In nitro-avidin, streptavidin-W120F, and -S45A, the reverse numbers of Ka are shown as Kd.b The pIs of streptavidin-DM3 and M4 were calculated from the full-length version of streptavidin (Met plus 159 amino acids), whereas those of the other streptavidin muteins were calculated from the core version.c The yield of the purified protein is shown; that of tamavidin 2-REV was calculated from 1 to 2 mg/50 mL.d NR, not reported.
2 f Biote
sc2
3s
mhTm(dadvf
bmibT2tp
StbdAbtttK
awbst
3t
tsttlwacffiatw
4 Y. Takakura et al. / Journal o
treptavidin-M4 (2.2 × 10−7M) whose biotin-bindings were almostompletely reversible (Laitinen et al., 2003; Wu and Wong, 2005,006).
.3. Subunit association, protease resistance, and thermaltability of tamavidin 2-REV
The subunit association of tamavidin 2-REV was analyzed byeans of gel-filtration and non-denaturing SDS-PAGE in which
eat denature of the protein prior to loading was omitted.he apparent molecular mass of tamavidin 2-REV was esti-ated at 53.4 kDa by means of gel-filtration chromatography
Fig. 4A) and at 60 kDa by means of SDS-PAGE under non-enaturing condition (Fig. 4B), respectively. On the other hand,
single band at 15.5 kDa was observed in SDS-PAGE underenaturing condition (Fig. 2B). These data suggest that the tama-idin 2-REV produced in E. coli is homo-tetramer in the nativeorm.
The tamavidin 2 tetramer was more stable after binding toiotin than in the absence of biotin and did not dissociate to theonomer even in SDS-PAGE (Fig. 5A). On the other hand, after bind-
ng to biotin, the monomer band was stronger than the tetramerand in the tamavidin 2-REV lane in the SDS-PAGE gel (Fig. 5B).hus, the inter-subunit associations of the tetrameric tamavidin-REV in the tamavidin 2-REV-biotin complex were weaker thanhose of the tetrameric tamavidin 2 in the tamavidin 2-biotin com-lex.
Proteins were incubated with protease K and separated byDS-PAGE to examine protease tolerance. Under these experimen-al conditions, BSA was completely degraded (Fig. 5C); however,oth tamavidin 2-REV and tamavidin 2 showed no signs of degra-ation in the presence or absence of biotin (Fig. 5A and B).lthough the tetramer band of tamavidin 2-REV incubated withiotin disappeared after protease K treatment, it is likely thathe tetramer only dissociated and was not degraded becausehe monomer band was intact (Fig. 5B). Our results thus showhat tamavidin 2-REV is as resistant as tamavidin 2 to protease.
With respect to the thermal stability, the Tm, the temperaturet which half of the protein was denatured, of tamavidin 2-REVas 80.9 ◦C, which was lower than the Tm of tamavidin 2 (86.9 ◦C),
ut comparable to that of streptavidin (79.9 ◦C). Thus, the thermaltability of tamavidin 2-REV was somewhat less stable than that ofamavidin 2 but as stable as that of streptavidin.
.4. Purification of a biotinylated protein by use of immobilizedamavidin 2-REV
Biotinylated BSA was chosen as a model substrate, and purifica-ion was attempted from a mixture of the substrate and the totaloluble proteins from an E. coli crude extract. The substrate alone orhe mixture was incubated with the tamavidin 2-REV immobilizedo the SepharoseTM resin. After the resin was washed, the biotiny-ated BSA was eluted by adding excess biotin, and the each fraction
as analyzed by use of SDS-PAGE (Fig. 6A and B). With the substratelone, the biotinylated BSA bound strongly to the resin because weould not detect protein in the unbound fraction or in the washraction (Fig. 6B). Then, the biotinylated BSA readily dissociatedrom the resin in the presence of excess free biotin at physiolog-
cal pH, with a recovery rate of 80% and a purity of 95% (Fig. 6And B). Thus, we successfully separated the model substrate fromhe crude protein preparation by means of affinity chromatographyith immobilized tamavidin 2-REV.
chnology 164 (2013) 19– 25
3.5. Comparison of tamavidin 2-REV with other modified proteinswith reversible biotin-binding capabilities
The characteristics of tamavidin 2-REV and other modifiedbiotin-binding proteins are summarized in Table 1. The advan-tages of tamavidin 2-REV over the other proteins listed in Table 1are quite evident. Nitro-avidin required a drastic pH change (pH4 for binding; pH 10 for elution) to purify a biotinylated protein,which may destabilize that protein. Moreover, the efficiency ofthe chemical modification was as low as 70% (Morag et al., 1996),necessitating pretreatment with free biotin to fill the unmodi-fied biotin-binding pockets. Reversible binding of a biotinylatedbiomolecule to avidin-W110K, monoavidin, streptavidin-W120F,and streptavidin-W120K has not been demonstrated. These pro-teins could not be produced in E. coli in soluble form. Insectcells were needed to produce monoavidin (Laitinen et al., 2003),avidin-W110K, and streptavidin-W120K (Laitinen et al., 1999).Streptavidin-W120F and streptavidin-S45A tend to accumulate ininclusion bodies in E. coli, and renaturation and tedious down-stream processing are required to prepare the active proteins (Hyreet al., 2000; Sano and Cantor, 1995). The lack of a cost-effectiveexpression and purification system for these modified proteins isprobably one of the reasons for their lack of successful practicalapplication for avidin/biotin technology. Recently, a streptavidinmutein with a modified loop7–8 has been developed (O’Sullivanet al., 2012). The tetrameric structure of this mutein was stabi-lized by a H127C mutation and the mutein showed an excellentreversible biotin-binding capability. However, a special strain ofBacillus subtilis and a special biotin-free medium were needed forproduction of this mutein, which are costly and require a high levelof technical expertise.
Monoavidin was reported to be protease-sensitive (Laitinenet al., 2003) and may not withstand incubation with crude cellextracts. Streptavidin-DM3 was the first monomeric streptavidinapplied to the purification of biotinylated protein; however, itshowed a strong tendency to aggregate because the monomeriza-tion exposed its hydrophobic residues at the surface (Qureshi andWong, 2002; Wu and Wong, 2006). On the other hand, anothermonomeric protein, streptavidin-M4, did not aggregate and wasexpressed at high levels in E. coli but was susceptible to a protease; itwas completely degraded after a 15-min incubation with proteaseK (Wu and Wong, 2005). The pI of tamavidin 2-REV was calculatedto 7.4 from the deduced amino acid sequence. This value is clearlylower than that of monoavidin, avidin-W110K, and streptavidin-M4, suggesting that the likelihood of charge-derived, non-specificbinding (Marttila et al., 2000) with tamavidin 2-REV should be lessthan that with these proteins. In the affinity chromatographies withstreptavidin-DM3 and streptavidin-M4, pretreatments of the crudecell extracts with dialysis to remove endogenous biotin, and withwild-type streptavidin to remove endogenous biotinylated pro-teins were performed before the purification of the biotinylatedproteins (Qureshi and Wong, 2002; Wu and Wong, 2006). Suchtreatments were not necessary in the present study. The recoveryrate of a biotinylated protein with tamavidin 2-REV (80%) was com-parable to that reported with streptavidin-M4 (70–80%, Wu andWong, 2006), but higher than that reported with streptavidin-S45A(50–60%, Malmstadt et al., 2003).
4. Conclusion
The remarkable reversible biotin-binding capability, protease
resistance, neutral pI, and high productivity of tamavidin 2-REVhighlight its value as a powerful tool for the purification of biotiny-lated biomolecules in studies of biochemistry, molecular biology,and biotechnology.
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cknowledgements
The authors are grateful to Dr. Yoshimitsu Kakuta, Kyushu Uni-ersity for helpful discussions regarding the crystallography of theamavidin 2-biotin complex. The authors acknowledge Mr. Kotarokajima, Sumika Chemical Analysis Service, Ltd. for helpful dis-ussions regarding the analysis using a Biacore biosensor, and Dr.oshihiko Komari for critical reading of the manuscript.
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