Site-specific tetrameric streptavidin-protein conjugation using sortase A

Journal of Biotechnology 152 (2011) 37–42

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Site-specific tetrameric streptavidin-protein conjugation using sortase A Takuya Matsumoto a , Shiori Sawamoto a , Takayuki Sakamoto a , Tsutomu Tanaka b,∗ , Hideki Fukuda b , Akihiko Kondo a a b

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan

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Article history: Received 1 September 2010 Received in revised form 7 January 2011 Accepted 12 January 2011 Available online 22 January 2011 Keywords: Streptavidin Sortase Modification Cold shock expression Site-specific

a b s t r a c t Streptavidin is tetrameric protein which has tight and specific biotin binding affinity, and streptavidin modification of proteins or small molecules is widely used for biotechnology tool. Here, we demonstrate site-specific streptavidin-protein conjugation using enzymes. We focused on sortase A, a transpeptidase from Staphylococcus aureus. A streptavidin-tagged LPETG motif (Stav-LPETG) was expressed in Escherichia coli. We achieved soluble streptavidin expression in E. coli without refolding using a cold shock expression system. Then we successfully conjugated Stav-LPETG with pentaglycine-appended green fluorescence protein (Gly5-GFP) or triglycine-appended glucose oxidase (Gly3-GOD) using sortase A. SDS-PAGE analysis showed site-specific tetrameric streptavidin-protein conjugation with the tagged proteins. In addition, the functions of a Stav-GOD conjugate, i.e., biotin-binding and glucose oxidase activity, were significantly higher compared to those of streptavidin-GOD conjugates prepared by chemical modification. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Streptavidin is a natural tetramer produced by Streptomyces avidinii that has tight and specific biotin binding affinity with a dissociation constant of about 10−15 M (Weber et al., 1989; Green, 1990; Grubmüller et al., 1996). By using its specific and tight biotin binding, streptavidin modification of other proteins or small molecules is widely used for biomolecule labeling, immobilization, and more sophisticated biotechnology applications (Ding et al., 2001; Hylarides and Mallett, 2001; Zhu et al., 2001; Caswell et al., 2003; Malmstadt et al., 2003; Ringler and Schulz, 2003; Howarth et al., 2006; Colonne et al., 2007; Choi et al., 2008; Suci et al., 2009; Lund et al., 2010). Recombinant streptavidin is usually expressed in E. coli (Green, 1990; Sano and Cantor, 1990, 1991) because recombinant expression in the natural host, S. avidinii, has not yet been established. However, the most serious problem is that streptavidin expressed in E. coli forms inclusion bodies and requires refolding (Sano and Cantor, 1990, 1991). Refolding approaches such as dilution, dialysis, and chromatography have been developed (Umetsu et al., 2003; Petrov et al., 2010), and hence refolded streptavidin can be successfully obtained (Gallizia et al., 1998; Sørensen et al., 2003). However, in the case of streptavidin fusion proteins, the refolding yield is significantly lower and a general refolding method has not been established partly because of tetrameric form of streptavidin. Consequently, many kinds of mod-

∗ Corresponding author. Tel.: +81 78 803 6202; fax: +81 78 803 6202. E-mail address: [email protected] (T. Tanaka). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.01.008

ified streptavidin have been prepared with chemical modification due to the limitations of streptavidin fusion protein preparation as described above. For example, alkaline phosphatase or horseradish peroxidase-conjugated streptavidin have been widely used for detection and quantification of target molecules (Bayer et al., 1986). Although chemical modification is simple and widely used to prepare streptavidin conjugates, the method often causes heterogeneity of products due to random modification. Other problems are the loss of protein functions or low yield of the products. Enzymatic approaches to site-specific protein modification have attracted much attention because the substrate specificity of an enzyme enables site-specific protein modification (Tanaka et al., 2005; Lin and Ting, 2006; Fontana et al., 2008). Several kinds of transglutaminases have been used for protein modification with primary amine containing molecules such as fluorescence probes (Lin and Ting, 2006) or PEG (Fontana et al., 2008). Briefly, a short substrate sequence for transglutaminase is genetically introduced at the N- or C-terminus of the target protein. The expressed protein is then modified with a small primary amine-containing molecule. The advantages of transglutaminase are high reaction efficiency and introduction of primary amine containing molecule into target proteins, however, one of the drawbacks is that substrate specificities have not been elucidated. In contrast, sortase has also been used for protein modification because of its high substrate specificities (Kruger et al., 2004). The most studied one is sortase A (SrtA), a transpeptidase from Staphylococcus aureus (Mazmanian et al., 1999; Novick, 2000; Paterson and Mitchell, 2004). SrtA recognizes the LPXTG sequence, cleaves between the Thr and Gly residues, and subsequently links the carboxyl group of Thr to an

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amino group of N-terminal glycine oligomers by a native peptide bond (Mazmanian et al., 1999; Novick, 2000). Recombinant soluble SrtA has been used for site-specific peptide–protein ligation in vitro (Mao et al., 2004). Recently, SrtA has also been used for protein–sugar (Smantaray et al., 2008) or protein–lipid ligation (Antos et al., 2008), and has been applied for protein immobilized beads preparation (Parthasarathy et al., 2007) or live cell imaging (Popp et al., 2007; Tanaka et al., 2008). The advantages of this enzymatic protein modification strategy are that it is highly selective and mild compared to conventional methods. In this study, we demonstrated site-specific tetrameric streptavidin conjugation using sortase A. We genetically introduced the LPETG sequence to the C-terminus of streptavidin (Stav-LPETG). As model partner proteins, pentaglycine-appended green fluorescence protein (Gly5-GFP) and triglycine-appended glucose oxidase (Gly3-GOD) were used. Interestingly, we succeeded in expressing the tagged streptavidin in a soluble form using an E. coli expression system without refolding. Soluble Stav-LPETG and Gly5-GFP (or Gly3-GOD) were site-specifically conjugated by SrtA-mediated ligation. Finally, we successfully detected target molecules using Stav-GFP and Stav-GOD conjugates. 2. Materials and methods 2.1. Construction of expression plasmids Ex-Taq DNA polymerase (Takara) was used for PCR, and all PCR-amplified sequences were verified by DNA sequencing. Expression plasmids for LPETG-tagged streptavidin at the Cterminus (Stav-LPETG) were constructed as follows. The gene encoding streptavidin was obtained by PCR using pWI3SAFlo318 as a template (Furukawa et al., 2006) using the 5 primer (5 -GGG GTA CCC TCG AGG CCG AGG CCG GCA TCA CCG GCA CCT GG-3 ) and the 3 primer (5 -GGA ATT CGG ATC CCG CTA GCC ACC AGT TTC CGG CAG AGA GCC ACC GGA GGC GGC GGA CGG CTT CAC CTT GGT GAA GGT-3 ). The amplified fragment was subcloned into the KpnI/EcoRI sites of the pCold I vector (Takara) to yield pColdIStav-LPETG. In E. coli, the plasmid expresses the protein construct TEE-His6-FactorXa cleavage site-streptavidin-LPETG (Qing et al., 2004). The plasmid for triglycine appended glucose oxidase (GOD) expression was constructed as follows. The gene encoding GOD was amplified by PCR using Aspergillus niger genomic DNA as a template using the following primers: 5 -GC CTC GAG AAA AGA GGC GGT GGA CAC CAC CAC CAC CAC CAC AGC AAT GGC ATT GAA GCC AGC CTC CTG ACT GAT CCC-3 , and 5 -G CAT AGC GGC CGC TCA CTG CAT GGA AGC ATA ATC TTC CAA-3 . The second PCR was carried out using the amplified fragment as a template using the following primers: 5 -G CAT ACT AGT AAA AGA GGC GGT GGA CAC CAC CAC CAC CAC CAC-3 , and 5 -G CAT AGC GGC CGC TCA CTG CAT GGA AGC ATA ATC TTC CAA-3 . The amplified fragment was ligated into the SpeI/NotI sites of pISI-EGFP (Adachi et al., 2008). The resultant plasmid was named pISI-Gly3-GOD. In Aspergillus. oryzae, the plasmid expresses the protein construct secretion signal-KR (cleavage site during secretion)-triglysine-GOD-His6. The N-terminal triglycine was exposed to protease cleavage during secretion into the culture medium (Adachi et al., 2008). 2.2. Expression and purification of Stav-LPETG The plasmid pColdI-Stav-LPETG was introduced into E. coli BL21 (DE3) pLysS. Cells were grown in LB medium to an OD (600 nm) of 0.5 at 37 ◦ C, then cells were incubated a further 30 min at 15 ◦ C. Expression of the protein was induced by the addition of isopropyl-␤-d-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After growth for an additional 24 h at 15 ◦ C, cells

were harvested by centrifugation (Qing et al., 2004). The cell pellets were resuspended in 50 mM phosphate, 150 mM NaCl, pH 8.0 and lysed by sonication. Stav-LPETG was purified from the soluble fraction by TALON metal affinity resin (Clontech) according to the manufacturer’s protocol, and dialyzed against 50 mM phosphate, 150 mM NaCl, pH 8.0. The concentration of purified Stav-LPETG was determined using a BCA protein assay kit (Pierce). 2.3. Expression and purification of Gly3-GOD, Gly5-GFP, and sortase A The expression and purification of Gly3-GOD was conducted as follows. The A. oryzae niaD mutant (strain IF4), derived from wildtype A. oryzae, OSI1031, was used as the GOD expression host. Czapek-Dox (CD) medium plates (2% glucose, 0.3% NaNO3 (CDNO3 ), 0.2% KCl, 0.1% KH2 PO4 , 0.05% MgSO4 ·7H2 O, and 0.8 M NaCl, pH 6.0) containing 1.5% agar were used as the minimal medium to select the fungal transformants. GPY medium (3% glucose, 0.2% KCl, 0.1% KH2 PO4 , 0.05% MgSO4 ·7H2 O, 1% peptone, and 0.5% yeast extract, pH 6.0) was used for growing the transformants and for GOD expression. The transformation of A. oryzae was carried out according to a previous report (Adachi et al., 2008). The resultant transformants were subcultured on CD-NO3 medium plates three times for obtaining stable expression transformants. Then the transformants were seeded in 250 ml of GPY medium and cultivated for 6 days at 30 ◦ C. The culture supernatants were separated using a Mira cloth (Millipore), and secreted GOD was purified using TALON metal affinity resin according to the manufacturer’s procedure. After purification, the protein concentration was determined as described above. Additionally, sortase A and Gly5-GFP were expressed and purified according to a previous report (Tanaka et al., 2008). 2.4. Transpeptidation reaction between Stav-LPETG and Gly3-GOD or Gly5-GFP The transpeptidation reaction was performed in 20 mM Tris–HCl, 150 mM NaCl, 0.5 mM CaCl2 , containing 10 ␮M His6 SrtA, 5 ␮M Stav-LPETG, and 20 ␮M Gly3-GOD (or Gly5-GFP) for 2 h at 37 ◦ C (pH 7.5). The reaction was terminated by mixing with SDS-PAGE sample buffer (50 mM Tris–HCl, 2% SDS, 6% 2mercaptoetahnol). The samples were then subjected to SDS-PAGE with or without boiling and the gels were stained with Coomassie brilliant blue R-250. 2.5. Conjugate activity assays Two kinds of streptavidin-GOD conjugates were prepared by two kinds of chemical modification. The streptavidinGOD conjugate was prepared by mixing 5 ␮M Stav-LPETG, 20 ␮M Gly3-GOD and 0.05% glutaraldehyde in reaction buffer (20 mM NaH2 PO4 , 150 mM NaCl, pH 7.5). Another chemical modification was performed with 0.2 M 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, hydrocholoride (EDC) (Dojindo), 0.05 M N-hydroxysuccinimide (NHS) (BACHEM), containing 5 ␮M Stav-LPETG and 20 ␮M Gly3-GOD for 2 h at ambient temperature (pH 7.5). Each conjugate prepared by SrtA or two kinds of chemical modifications was incubated in a 96-well biotin coated polystyrene plate (Pierce) for 30 min at 4 ◦ C. The wells were then washed with PBS twice, and GOD activity was assayed with an ELISA POD substrate TMB Kit (Nacalai Tesque) according to the manufacturer’s protocol. Then the absorbance of 450 nm was analyzed with a plate reader (Wallac 1420 ARVOsx). The amount of streptavidin immobilized on biotin coated plate was also determined. Each streptavidin-GOD conjugate was incuvated in 96-well biotin coated plate for 30 min at 4 ◦ C. The wells were

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Fig. 1. SDS-PAGE analysis of LPETG-tagged streptavidin after imidazole elution from His-tag affinity resin. (A) Without boiling of samples, (B) with boiling of samples. Lanes 1–4 show fractions after imidazole elution (fraction volume is about 1 ml). The volume of loaded samples were 10 ␮L.

then washed with PBS twice, and 0.1 ␮g anti-streptavidin mouse monoclonal antibody (abcam) per well was incubated for 1 hour at room temperature. Then the wells were also washed with PBS and 0.1 ␮g HRP conjugated anti-mouse antibody (promega) per well was incubated for 1 h at room temperature, after washing well with PBS HRP activity was assayed with an ELISA POD substrate TMB Kit. Then the absorbance of 450 nm was analyzed with a plate reader (Wallac 1420 ARVOsx). 3. Results and discussion 3.1. Expression of tagged streptavidin in a soluble form using E. coli as a host First, we tried to express streptavidin as an inclusion body according to various previous reports (Sano and Cantor, 1990, 1991). Using a T7 promoter expression system, a large amount of inclusion body was obtained; however, optimizing the tagged streptavidin refolding process was relatively difficult (data not shown). Therefore, we employed a cold shock vector expression system (Qing et al., 2004), which drives the high expression of cloned genes upon induction by cold shock. We introduced LPETGtagged streptavidin into a pCold vector and tagged streptavidin was expressed using E. coli BL21 (DE3) as a host. After IPTG induction, the streptavidin was purified from the soluble fraction of cell lysates using TALON affinity resin. Fig. 1 shows SDS-PAGE analysis of the purified streptavidin after imidazole elution from TALON affinity resin. When using purified and recombinant streptavidin without boiling before SDS-PAGE, a band corresponding to streptavidin was observed around 60 kDa, which is in accordance with a previous report (Howarth et al., 2006) (Fig. 1A). From SDS-PAGE of boiled samples, the streptavidin tetramer was dissociated and a band corresponding to streptavidin monomers was observed around 16 kDa (Fig. 1B). This clearly showed that a large amount of streptavidin was successfully expressed and easily purified in a soluble form using an E. coli expression host. The yield of streptavidin was about 10 mg/L. 3.2. Site-specific streptavidin-protein conjugation using sortase We investigated the conjugation reaction between Stav-LPETG and triglycine-appended functional proteins using SrtA. Enhanced green fluorescent protein (GFP) and glucose oxidase (GOD) were

Fig. 2. SDS-PAGE analysis of the site-specific reaction products after sortase A treatment. Each samples were boiled before SDS-PAGE analysis (A) lane 1, StavLPETG; lane 2, Gly5-GFP; lane 3, sortase A; lane 4, Stav-LPETG and Gly5-GFP; lane 5, Stav-LPETG and sortase A; lane 6, Gly5-GFP and sortase A; lane 7, Gly5-GFP and Stav-LPETG and sortase A. (B) Lane 1, Stav-LPETG; lane 2, Gly3-GOD; lane 3, sortase A; lane 4, Stav-LPETG and Gly3-GOD; lane 5, Stav-LPETG and sortase A; lane 6, Gly3-GOD and sortase A; lane 7, Gly3-GOD and Stav-LPETG and sortase A.

used as model proteins. The substrate sequence of SrtA, pentaglysine and triglysine was genetically introduced into the N-termini of GFP (Gly5-GFP) and GOD (Gly3-GOD). Tagged-GFP was expressed using E. coli and tagged-GOD was expressed using A. oryzae as a host (Adachi et al., 2008). Fig. 2A shows SDS-PAGE analysis after the transpeptidation reaction between Gly5-GFP and Stav-LPETG using SrtA. To enhance transpeptidation reaction, high SrtA concentration (more than 10 ␮M) was required (data not shown). All samples were analyzed after boiling to confirm conjugation through covalent bonds between streptavidin monomers and Gly5-GFP. When all substrates, Stav-LPETG (16 kDa), Gly5-GFP (27 kDa), and SrtA, were mixed, a new band appeared around 43 kDa (Fig. 2A, lane 7), which corresponds to the streptavidin-GFP conjugate. Alternatively, no band was observed from a mixture of Gly5-GFP and Stav-LPETG without SrtA (Fig. 2A, lane 6). In addition, no conjugate was observed from a mixture of streptavidin without the LPETG tag and Gly5-GFP in spite of the presence of SrtA (data not shown). These results show site-specific modification between Strav-LPETG and Gly5-GFP using SrtA. Similar results were obtained with Gly3-GOD as the partner protein. When Stav-LPETG, Gly3-GOD, and SrtA were mixed, a new band appeared around 120 kDa (Fig. 2B, lane 7), whose size corresponds to the streptavidin-GOD conjugate. The yield of streptavidin-GFP or streptavidin-GOD was about 60% of Stav-LPETG as evaluated by the ratio of the Stav-LPETG band intensity before conjugation (Fig. 2, lane 6) to that after conjugation (Fig. 2, lane 7), which suggesting the number of glycine residues at N-terminal does not contribute significantly to the transpeptidation reaction (Huang et al., 2003) and both triglycine and pentaglycine are utilizable for SrtA-mediated protein conjugation. The yield corresponded to the value of our previous report, the protein–protein conjugation

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(Sakamoto et al., 2010). One of the possibilities of the lower conjugation efficiency is steric hindrance between streptavidin and protein, because in the case of protein modification with small molecule such as synthesized fluorescence probe, higher yield was obtained in previous reports (Huang et al., 2003; Kruger et al., 2004; Mao et al., 2004; Parthasarathy et al., 2007; Popp et al., 2007). The band appeared at around 30 kDa on lane4 in Fig. 2A and B was assumed to be streptavidin dimer conjugated by SrtA, because SrtA has relatively broader substrate specificity for N-terminal polyglycine residues (Huang et al., 2003). Although the amino acid sequence at N-termini of our recombinant streptavidin is Asp-His-Lys-Val-(His)6 , no report tried to carry out the conjugation between LPETG motif and N-terminal asparagine residue, implying further studies will be required about the substrate specificity of sortase A reaction. Alternatively, streptavidin dimers were observed only in the mixture of streptavidin and SrtA without appropriate nucleophile (e.g. Gly5-GFP or Gly3-GOD), no streptavidin dimer was produced in the presence of Gly5-GFP, streptavidin and SrtA (Fig. 2A). Meanwhile, Fig. 2B shows streptavidin dimer was observed in spite of the presence of Gly3-GOD. One possible explanation is that GOD might cause steric hindrance during sortase A conjugation reaction with streptavidin because of the large size of GOD, which is partially supported by the fact that the band was slightly decreased in the presence of excess amount of GOD (data not shown). We also determined the number of target proteins tethered to tetrameric streptavidin. The tetramers could be distinguished by SDS-PAGE, if the samples were not boiled (Howarth et al., 2006), according to the number of tethered target protein (e.g. GFP or GOD). Fig. 3 shows that one target protein (e.g. GFP or GOD) was tethered to tetrameric streptavidin (Fig. 3A and B, lane7), suggesting steric hindrance inhibited to tether more numbers of target proteins to tetrameric streptavidin. These results demonstrated SrtA-mediated site-specific streptavidin conjugation with functional proteins. 3.3. Evaluation of biotin binding ability and enzymatic activity of a streptavidin-protein conjugate We also investigated the function of a streptavidin-protein conjugate prepared with the SrtA-mediated reaction. From Fig. 4, GOD activity after SrtA reaction was retained as almost same those of GOD or the mixture of GOD and streptavidin, suggesting SrtA reaction did not affect on the protein function. Similar results were obtained in the case of Gly5-GFP, i.e. without loss of fluorescence (data not shown). Then we utilized streptavidin-GOD conjugate to evaluate its bifunctionality, because it is ease to detect the amplified signals through GOD activity. The streptavidin-GOD conjugate was incubated in a biotincoated polystyrene plate and the unbound moiety was washed out. Then GOD activity was assayed. When Stav-LPETG was conjugated with Gly3-GOD using SrtA, the streptavidin-GOD conjugate had both biotin binding affinity and GOD activity; therefore, GOD activity was preserved (Fig. 5, column 7). On the other hand, without SrtA, Stav-LPETG was not conjugated with Gly3-GOD and no GOD activity was detected (Fig. 5, column 6). In addition, no GOD activity was detected when streptavidin without the LPETG tag was used for conjugation (data not shown). These results show that SrtA enabled conjugation between tagged streptavidin and tagged functional proteins (Fig. 6). 3.4. Functional comparison of conjugates by chemical modification and SrtA modification One of the advantages of enzyme-mediated protein conjugation is site-specific conjugation without the loss of function. We

Fig. 3. SDS-PAGE analysis using polyacrylamide-gradient gel of the site-specific reaction products after sortase A treatment. Each samples were analyzed by SDSPAGE without boiling. (A) Lane 1, Stav-LPETG; lane 2, Gly5-GFP; lane 3, sortase A; lane 4, Stav-LPETG and Gly5-GFP; lane 5, Stav-LPETG and sortase A; lane 6, Gly5-GFP and sortase A; lane 7, Gly5-GFP and Stav-LPETG and sortase A. (B) Lane 1, StavLPETG; lane 2, Gly3-GOD; lane 3, sortase A; lane 4, Stav-LPETG and Gly3-GOD; lane 5, Stav-LPETG and sortase A; lane 6, Gly3-GOD and sortase A; lane 7, Gly3-GOD and Stav-LPETG and sortase A.

thus compared the activities of the streptavidin-GOD conjugate prepared by SrtA-mediated conjugation and a Stav-GOD conjugate prepared by chemical modification. Crosslinking between Stav-LPETG and Gly3-GOD was achieved with glutaraldehyde or EDC/NHS reaction. In the case of Streptavdin-GOD conjugate prepared by SrtA reaction, Stav-LPETG (5 ␮M) and Gly3-GOD (20 ␮M)

Fig. 4. Activity of GOD before and after modification of sortase A. Left column; GOD. Middle column; the mixture of GOD and streptavidin (before modification of sortase A) right column; the mixture of GOD, streptavidin and sortase A (after modification of sortase A). The GOD concentration in all samples was 20 ␮M.

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jugate immobilized on biotin coated plate using anti-streptavidin antibody. The amount of immobilized streptavidin-GOD conjugate prepared by SrtA modification was about 2-fold higher than that of the conjugate prepared by chemical modification (data not shown). This result suggests chemical modification also may have caused the loss of biotin binding ability. In conclusion, we demonstrated SrtA-mediated streptavidinprotein conjugation. Streptavidin with a short peptide tag introduced was expressed in a soluble form using a cold shock expression system. Then the tagged streptavidin was sitespecifically conjugated with tagged partner proteins that retained their functions. Our study is first time as site-specific multimeric protein modification using sortase, and our results show a promising way to expand the use of streptavidin conjugates. Conflicts of interest We declare no conflicts of interest.

Fig. 5. Evaluation of biotin binding and GOD activity after sortase treatment. Column 1, Stav-LPETG; Column 2, Gly3-GOD; Column 3, sortase A; Column 4, Stav-LPETG and sortaseA; Column 5, Gly3-GOD and sortase A; Column 6, Stav-LPETG and Gly3-GOD; Column 7, Gly3-GOD and Stav-LPETG and sortase A.

were mixed and SrtA was added. In the case of streptavidin-GOD conjugate prepared by glutaraldehyde or EDC/NHS crosslinking, Stav-LPETG (5 ␮M) and Gly3-GOD (20 ␮M) were mixed and 0.05% glutaraldehyde or EDC (0.2 M)/NHS (0.05 M) was added. The biotinbinding activity and GOD activity of each conjugate was analyzed as described above. The GOD activity after SrtA reaction was retained as almost same those of GOD or the mixture of GOD described above. Meanwhile, the GOD activities reduced to about 80% in the case of glutaraldehyde modification or about 60% in EDC/NHS modification. In addition, the streptavidin-GOD conjugate prepared by SrtA conjugation showed higher biotin binding ability and higher GOD activity than the conjugate prepared by chemical modification (Fig. 4). SDS-PAGE analysis showed a number of high molecular mass bands in the case of glutaraldehyde or EDC/NHS crosslinking (data not shown), which suggests that random modification may have caused the loss of biotin binding activity and/or GOD activity. We also determined the amount of streptavidin-GOD con-

Fig. 6. GOD activity of the streptavidin-GOD conjugate after binding to a biotincoated plate. Left column; streptavidin-GOD conjugate prepared by sortase A reaction. Middle column; streptavidin-GOD conjugate prepared by glutaraldehyde. Right column; streptavidin-GOD conjugate prepared by EDC/NHS reaction.

Acknowledgements This work was supported in part by a Grant-in-Aid for Young Scientist B (21760638) of Japan Society for the Promotion of Science (JSPS) and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), MEXT, Japan. References Adachi, T., Ito, J., Kawata, K., Kaya, M., Ishida, H., Sahara, H., Hata, Y., Ogino, C., Fukuda, H., Kondo, A., 2008. Construction of an Aspergillus oryzae cell-surface display system using a putative GPI-anchored protein. Appl. Microbiol. Biotechnol. 81, 711–719. Antos, J.M., Miller, G.M., Grotenbreg, G.M., Ploegh, H.L., 2008. Lipid modification of proteins through sortase-catalyzed transpeptidation. J. Am. Chem. Soc. 130, 16338–16343. Bayer, E.A., Ben-Hur, H., Wilchek, M., 1986. A sensitive enzyme assay for biotin, avidin, and streptavidin. Anal. Biochem. 154, 367–370. Caswell, K.K., Wilson, J.N., Bunz, U.H.F., Murphy, C., 2003. Preferential end-to-end assembly of gold nanorods by biotin–streptavidin connectors. J. Am. Chem. Soc. 125, 13914–13915. Choi, J., Wang, N.S., Reipa, V., 2008. Conjugation of the photoluminescent silicon nanoparticles to streptavidin. Bioconjug. Chem. 19, 680–685. Colonne, M., Chen, Y., Wu, K., Freiberg, S., Giasson, S., Zhu, X.X., 2007. Binding of streptavidin with biotinylated thermosensitive nanospheres based on poly (N,N-diethylacrylamide-co-2-hydroxyethyl methacrylate). Bioconjug. Chem. 18, 999–1003. Ding, Z., Fong, R.B., Long, C.J., Stayton, P.S., Hoffman, A.S., 2001. Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield. Nature 411, 59–62. Fontana, A., Spolaore, B., Mero, A., Veronese, F.M., 2008. Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv. Drug Deliv. Rev. 60, 13–28. Furukawa, H., Tanino, T., Fukuda, H., Kondo, A., 2006. Display system for homooligomeric protein by coexpression of native and anchored subunits. Biotechnol. Prog. 22, 994–997. Gallizia, A., de Lalla, C., Nardone, E., Santambrogio, P., Brandazza, A., Sidoli, A., Arosio, P., 1998. Production of soluble and functional recombinant streptavidin in Escherichia coli. Protein. Expr. Purif. 14, 192–196. Green, N.M., 1990. Avidin and Streptavidin. Methods Enzymol. 184, 51–67. Grubmüller, H., Heymann, B., Tavan, P., 1996. Ligand binding: molecular mechanics calculation of the streptavidin-biotin rupture force. Science 271, 997–999. Howarth, M., Chinnapen, D.J., Gerrow, K., Dorrestein, P.C., Grandy, M.R., Kelleher, N.L., El-Husseini, A., Ting, A.Y., 2006. A monovalent streptavidin with single femtomolar biotin binding site. Nat. Methods 3, 267–273. Huang, X., Aulabaugh, A., Ding, W., Kapoor, B., Alksne, L., Tabei, K., Ellestad, G., 2003. Kinetic mechanism of Staphylococcus aureus sortase SrtA. Biochemistry 42, 11307–11315. Hylarides, M.D., Mallett, R.W., 2001. A robust method for the preparation and purification of antibody/streptavidin conjugates. Bioconjug. Chem. 12, 421–427. Kruger, R.G., Otvos, B., Frankel, B.A., Bentley, M., Dostal, P., McCafferty, D.G., 2004. Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA. Biochemistry 43, 1541–1551. Lin, C.W., Ting, A.Y., 2006. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. J. Am. Chem. Soc. 128, 4542–4543.

42

T. Matsumoto et al. / Journal of Biotechnology 152 (2011) 37–42

Lund, K., Manzo, A.J., Dabby, N., Michelotti, N., Johnson-Buck, A., Nangreave, J., Taylor, S., Pei, R., Stojanovic, M.N., Walter, N.G., Winfree, E., Yan, H., 2010. Molecular robots guided by prescriptive landscapes. Nature 465, 206–210. Malmstadt, N., Hyre, D.E., Ding, Z.L., Hoffman, A.S., Stayton, P.S., 2003. Affinity thermoprecipitation and recovery of biotinylated biomolecules via a mutant streptavidin – smart polymer conjugate. Bioconjug. Chem. 14, 575–580. Mao, H., Hart, S.A., Schink, A., Pollok, B.A., 2004. Sortase-mediated protein ligation: a new method for protein engineering. J. Am. Chem. Soc. 126, 2670–2671. Mazmanian, S.K., Liu, G., Ton-That, H., Schneewind, O., 1999. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285, 760–763. Novick, R.P., 2000. Sortase: the surface protein anchoring transpeptidase and the LPXTG motif. Trends. Microbiol. 8, 148–151. Parthasarathy, R., Subramanian, S., Boder, E.T., 2007. Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug. Chem. 18, 469–476. Paterson, G.K., Mitchell, T.J., 2004. The biology of Gram-positive sortase enzymes. Trends. Microbiol. 12, 89–95. Petrov, S., Nacheva, G., Ivanov, I., 2010. Purification and refolding of recombinant human interferon-gamma in urea–ammonium chloride solution. Protein. Expr. Purif. 73, 70–73. Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E., Ploegh, H.L., 2007. Sortagging: a versatile method for protein labeling. Nat. Chem. Biol. 3, 707–708. Qing, G., Ma, L.C., Khorchid, A., Swapna, G.V., Mal, T.K., Takayama, M.M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M., Inouye, M., 2004. Coldshock induced high-yield protein production in Escherichia coli. Nat. Biotechnol. 22, 877–882. Ringler, P., Schulz, G.E., 2003. Self-assembly of proteins into designed networks. Science 302, 106–109.

Sakamoto, T., Sawamoto, S., Tanaka, T., Fukuda, H., Kondo, A., 2010. Enzymemediated site-specific antibody-protein modification using a ZZ domain as a linker. Bioconjug. Chem. 21, 2227–2233. Sano, T., Cantor, C.R., 1990. Expression of a cloned streptavidin gene in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 87, 142–146. Sano, T., Cantor, C.R., 1991. Expression vectors for streptavidin-containing chimeric proteins. Biochem. Biophys. Res. Commun. 176, 571–577. Smantaray, S., Marathe, U., Dasgupta, S., Nandicori, V.K., Roy, R.P., 2008. Peptidesugar ligation catalyzed by transpeptidase Sortase: A facile approach to neoglycoconjugate synthesis. J. Am. Chem. Soc. 130, 2132–2133. Sørensen, H.P., S-Petersen, H.U., Mortensen, K.K., 2003. Dialysis strategies for protein refolding: preparative streptavidin production. Protein. Expr. Purif. 31, 149–154. Suci, P.A., Kang, S., Young, M., Douglas, T., 2009. A Streptavidin-protein cage janus particle for polarized targeting and modular functionalization. J. Am. Chem. Soc. 131, 9164–9165. Tanaka, T., Kamiya, N., Nagamune, T., 2005. N-terminal glycine-specific conjugation catalyzed by microbial transglutaminase. FEBS Lett. 579, 2092–2096. Tanaka, T., Yamamoto, T., Tsukiji, S., Nagamune, T., 2008. Site-specific protein modification on living cells catalyzed by sortase. ChemBioChem. 9, 802–807. Umetsu, M., Tsumoto, K., Hara, M., Ashish, H., Goda, S., Adschiri, T., Kumagai, I., 2003. How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system. J. Biol. Chem. 278, 8979–8987. Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J., Salemme, F.R., 1989. Structural origins of high-affinity biotin binding to streptavidin. Science 243, 85–88. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., Snyder, M., 2001. Global analysis of protein activities using proteome chips. Science 293, 2101–2105.