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Enzyme and Microbial Technology 48 (2011) 307–311

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

utodisplay of streptavidin

in Parka, Joachim Joseb, Sarah Thömmesb, Jo-Il Kima, Min-Jung Kangc, Jae-Chul Pyuna,∗

School of Materials and Sciences, College of Engineering, Yonsei University, 134 Shin-chon-dong, Seo-dae-mun-gu, Seoul 120-749, Republic of KoreaInstitute of Pharmaceutical Chemistry, Heinrich Heine University, Duesseldorf, GermanyKorea Institute of Science and Technology (KIST), Seoul, Republic of Korea

r t i c l e i n f o

rticle history:eceived 19 June 2010eceived in revised form 31 October 2010

a b s t r a c t

Streptavidin was expressed on the outer membrane of E. coli as a recombinant fusion protein with anautotransporter domain called AIDA-I (adhesin involved in diffuse adherence) using autodisplay technol-

ccepted 11 December 2010

eywords:utodisplaytreptavidin. coli

ogy. The autodisplay of streptavidin was confirmed by SDS-PAGE of the outer membrane proteins, and thenumber of autodisplayed streptavidin molecules on a single E. coli cell was evaluated with densitometricanalysis. The biotin-binding activity of the autodisplayed streptavidin was estimated after treatment withfluorescently labeled biotin by fluorescence microscopy and flow cytometry. The biotin-binding activityof the E. coli with autodisplayed streptavidin was compared with the activity of streptavidin immobilizedon magnetic beads. Finally, the outer membrane presenting autodisplayed streptavidin was isolated and

ropla

mmunoassay layered on a 96-well mic

. Introduction

Autodisplay technology is a method for the surface display ofroteins. In this paper, autodisplay technology was used to expressroteins or peptides fused with an autotransporter domain calledIDA-I (adhesin involved in diffuse adherence) from E. coli [1].s shown in Fig. 1, the recombinant fusion protein was made by

ntroducing the coding sequence of the passenger protein in-frameetween the signal peptide and the translocation domain of a trans-ormation vector [2,3]. The C-terminal part of the autotransporterrotein forms a porin-like structure (�-barrel) within the outerembrane of Gram-negative bacteria, and the recombinant pas-

enger domain is translocated to the surface through this pore4–7]. Such an autodisplay system has been reported to express

ore than 105 recombinant molecules on the outer membrane ofsingle E. coli cell [8].

Recently, we reported the autodisplay of Z-domains, which arene of the IgG binding domains of Protein A from S. aureus [9]. Ashe Z-domain binds specifically to the polysaccharides at the con-tant (Fc) region of antibodies, it has been applied to immunoassaysor the control of antibody orientation [10–15]. The E. coli withutodisplayed Z-domains were reported to be applicable for sig-

al amplification of the surface plasmon resonance (SPR) biosensor9]. To achieve this signal amplification, antibody-bound E. coli cellseacted to antigens that were already bound to antibodies on thePR biosensor surface. The E. coli cells presenting Z-domains could

∗ Corresponding author. Tel.: +82 2 2123 5851; fax: +82 2 365 5882.E-mail address: jcpyun@yonsei.ac.kr (J.-C. Pyun).

141-0229/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2010.12.006

te for an immunoassay.© 2010 Elsevier Inc. All rights reserved.

also be used as a solid support for highly sensitive immunoassays[16]. The outer membrane of E. coli, with autodisplayed Z-domainsisolated and layered on a 96-well plate, has been used to develop ahypersensitive sandwich-type immunoassay [17]. In our previouswork, the low non-specific binding of proteins to the outer mem-brane of E. coli was reported to contribute to the improvement of thesensitivity of this technique [17]. The outer membrane can also belayered on various surfaces via hydrophobic interactions [16,17].Such an outer membrane layer can also be prepared on the goldsurface of an SPR biosensor for the preparation of a highly sensitivemolecular recognition layer [18].

In this work, the generation of cells that autodisplay strep-tavidin is presented. Streptavidin is a tetrameric biotin bindingprotein with biotin binding sites at each subunit. Streptavidin hasan unusually high affinity for biotin (Kd = 1013–1016 M), and thisnon-covalent interaction is considered to be almost irreversible[19,20]. Because of such tight binding characteristics, streptavidinhas been used for many in vitro and in vivo applications [21–25].Here the autodisplay of streptavidin was confirmed by SDS-PAGEanalysis of outer membrane proteins. The number of autodisplayedstreptavidin molecules on a single E. coli cell was evaluated withdensitometric analysis, and the results were compared with a ref-erence protein of the E. coli outer membrane called OmpA.

The biotin-binding activity of the autodisplayed streptavidinwas estimated after treatment with fluorescently labeled biotin by

fluorescence microscopy and flow cytometry analysis. The biotin-binding activity of the E. coli with autodisplayed streptavidinwas compared to similarly sized magnetic beads with covalentlyimmobilized streptavidin. The outer membrane with autodisplayedstreptavidin was isolated and layered on a 96-well microplate, and

308 M. Park et al. / Enzyme and Microbial Technology 48 (2011) 307–311

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Fig. 2. SDS-PAGE analysis of outer membrane proteins. Lane M indicates the molec-ular weight marker. Lane 1 and lane 2 represent the outer membrane proteins from

Fig. 1. The pST001 autodisplay vector of streptavidin.

he biotin-binding activity was estimated to determine its applica-ility in an immunoassay.

. Materials and methods

.1. Construction of a plasmid for the surface display of streptavidin

The streptavidin gene was presented by Dr. S.J. Chung (KRIBB, Korea). Thetreptavidin gene from the pET-28a plasmid was amplified by PCR, and primersith adhesive restriction sites for XhoI and KpnI were used to make the following

equences: (forward) 5′-CTC GAG GAC CCC TCC AAG GAC TCG AAG-3′ and (reverse)′-GGT ACC CTG CTG AAC GGC GTC GAG CG-3′ (Sigma Aldrich, Munich, Germany).he PCR product (487 base pairs) was first inserted into the TOPO 4.0 vector (Invit-ogen, Karlsruhe, Germany) according to the manufacturer’s instructions. Then thensert was excised by KpnI and XhoI (New England Biolabs, Frankfurt, Germany),

hich was confirmed by agarose gel electrophoresis. The fragments encoding strep-avidin were extracted from the gel (Qiaquick Gel Extraction Kit, Qiagen, Hilden,ermany) and then ligated into the pET-ADX-04 plasmid, which was cut by XhoI andpnI [26]. To avoid religation of pET-ADX-04, BglII was added to the ligation mixture

o cut the gene fragment encoding Adx. The final plasmid was verified through DNAequence analysis and named pST001.

.2. Surface display of streptavidin

The plasmid pST001 was transformed into E. coli UT5600 (DE3) (F-, ara-14, leuB6,ecA6, lacY1, proC14, tsx-67, � (ompT-fepC) 266, entA403, trpE38, rfbD1, rpsL109Strr), xyl-5, mtl-1, thi-1) [26] by electroporation. E. coli UT5600 (DE3) pST001 cellsere grown overnight at 37 ◦C and continuously shaken (200 rpm) in lysogeny brothedium (LB medium, 10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter) con-

aining 50 mg/l carbenicillin. Cells were grown until the optical density (OD) reached.5 (578 nm). Protein expression was induced by the addition of 1 mM IPTG andubsequent incubation for 1 h at 30 ◦C with vigorous shaking (200 rpm) [2].

.3. Flow cytometry analysis

E. coli UT5600 (DE3) pST001 and E. coli UT5600 (DE3) were routinely grown asescribed above. Protein expression started at an E. coli concentration of OD578 nm

.3–0.4, and 1 ml of the culture was harvested and submitted to the follow-ng washing steps: (1) 5% bovine serum albumin (BSA) in PBS and (2) washingwice with PBS. The cells were the resuspended in 100 �l phosphate-bufferedaline (PBS), and a 15 mM biotinylated fluorescein solution (2 �l) was added.fter repeating the washing steps twice with PBS, the solutions were filteredith a 0.22-�m filter. For each FACS analysis, 50,000 cells were analyzed with aow cytometer at the excitation wavelength of 496 nm (Cyflow, Partec, Münster,ermany).

.4. Outer membrane preparation

The outer membranes of E. coli cells were prepared as described in our previousork [9]. E. coli cells were grown overnight, and the culture (1 ml) was used to inoc-late fresh LB medium (20 ml). The E. coli cells were incubated at 37 ◦C with vigoroushaking (200 rpm) for approximately 5 hr until the OD578 nm reached 0.5. Then the. coli cells were harvested, and the outer membranes were prepared following theapid isolation method of Hantke [27] using modifications by Schulthesis et al. [28].

.5. SDS-PAGE and densitometric analysis

The outer membrane proteins were analyzed with 12.5% SDS-PAGE [15].fter electrophoresis, proteins were visualized by staining with Coomassie bril-

iant blue. The density of each band was estimated using a documentationystem (ChemiDoc XRSTM) and an analysis program (Quantity-OneTM) from BioRadaboratories.

UT5600(DE3) without autodisplayed streptavidin and the outer membrane proteinsfrom UT5600(DE3) transformed with pST001 for the autodisplay of streptavidin,respectively.

2.6. Preparation and activity assay of streptavidin-coated magnetic beads

Amine-modified magnetic beads with a 1-�m diameter were used to covalentlyimmobilize streptavidin through a reaction with glutaraldehyde [33]. Differentamounts of streptavidin were immobilized on the magnetic beads by treatmentwith streptavidin solutions of different concentrations. After blocking with BSA(10 mg/ml), the magnetic beads were treated with 1 �g/ml biotinylated horseradishperoxidase (HRP). To quantify the bound biotinylated HRP, the beads were treatedwith the chromogenic substrate (TMB), and 2 M sulfuric acid was used to quenchthe HRP reaction.

3. Results and discussion

3.1. Autodisplay of streptavidin

The protein band of autodisplayed streptavidin, with a molecu-lar weight of 69.2 kDa, was observed, as shown in Fig. 2. As expectedfrom the specification of the E. coli strain UT5600 (F− ara14 leuB6azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1thi1, �ompT-fepC266), the protein band correlating to autodis-played streptavidin did not appear, as shown in lane 1. In the caseof the UT5600(DE3) strain transformed with the autodisplay vec-tor pST001, the protein band of autodisplayed streptavidin, with amolecular weight of 69.2 kDa, was observed, as shown in Lane 2.As the expression level of the outer membrane protein OmpA ofthe E. coli strain UT5600 is known to be approximately 105 copiesper E. coli cell [29], the number of the autodisplayed streptavidinmolecules was evaluated by normalizing the intensity of the pro-tein bands in the SDS-PAGE gel. From the densitometric calculation,the number of streptavidin molecules per E. coli cell was estimatedto be 1.6 × 105 molecules/cell [9,29–32].

The biotin-binding activity of autodisplayed streptavidin wastested by treatment with biotinylated fluorescein. An E. coliculture with an OD578 1.0 (200 �l) was treated with 100 g/ml(100 l) biotinylated fluorescein. Ten microliters of the biotinylatedE. coli solution was selected for use after a 10-fold dilution withPBS. As shown in Fig. 3, E. coli with autodisplayed streptavidinshowed intense fluorescence signals, which indicated the bind-

ing of biotinylated fluorescein. Intact E. coli cells, however, showedno significant fluorescence signal under the fluorescence micro-scope. This result indicates that the autodisplayed streptavidin onthe outer membrane of E. coli has biotin-binding activity.

M. Park et al. / Enzyme and Microbial Technology 48 (2011) 307–311 309

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ig. 3. Activity test of the autodisplayed streptavidin with treatment of biotinylatednd UT5600(DE3) pST001 cells treated with biotinylated fluorescein.

The E. coli with autodisplayed streptavidin and the intact E. coliT5600 were also stained with biotinylated fluorescein to test

he biotin-binding activity of autodisplayed streptavidin by flowytometry. As shown in Fig. 3, the intact E. coli cells were observedt a low fluorescence signal range. On the other hand, most E. coliells with autodisplayed streptavidin were observed at a higher flu-rescence signal range than that of the intact E. coli cells. The ratiof the intact E. coli and the E. coli with autodisplayed streptavidinver the level of 10 (arbitrary unit) was estimated to be less than 5%nd more than 95% of the total number of E. coli cells, respectively.hese results indicate that the yield of E. coli with autodisplayedtreptavidin was more than 95% (Fig. 4).

.2. Activity of E. coli outer membrane layer with autodisplayedtreptavidin

The applicability of E. coli cells with the autodisplayed strep-avidin as a solid support for immunoassays was tested. Theiotin-binding activity of autodisplayed streptavidin on the outerembrane of E. coli was compared with the streptavidin-coated

-�m diameter magnetic particles. The magnetic beads with anmino group on the surface were used for covalent immobiliza-ion of streptavidin by reacting them with glutaraldehyde [33]. As

hown in Fig. 5(a), the maximum activity of the streptavidin-coatedagnetic beads was obtained by an immobilization reaction with

he streptavidin solution at 80–90 ng/ml.The E. coli cells with autodisplayed streptavidin were compared

ith the streptavidin-coated magnetic beads. The quantity of E.

ig. 4. Flow cytometric analysis of the biotin-binding activity. The E. coli transformed wuorescein.

escein. The phase-contrast and fluorescence images were taken from UT5600(DE3)

coli was adjusted such that the total surface area was the sameas that of the magnetic beads selected for comparative analysis.The E. coli cell was assumed to have a cylindrical structure with alength of 2 �m and a diameter of 1 �m [34]. In this case, the sur-face area of a single E. coli cell was calculated to be 7.85 �m2. Foreach measurement, approximately 120 �l of E. coli culture at anOD578 of 1.0 was used, which corresponds to the surface area ofa single well of a 96-well microplate (flat bottom) with 100 �l ofan aqueous solution. As shown in Fig. 5(b), the assay curve of E.coli with the autodisplayed streptavidin reached saturation with300 ng/ml biotinylated HRP. The magnetic beads with the maxi-mum biotin-binding activity (•) showed saturation with 100 ng/mlbiotinylated-HRP. This result implies that the E. coli with autodis-played streptavidin have a surface concentration of streptavidinat least three times greater than that of the magnetic beads withthe maximum surface concentration of streptavidin, as shown inFig. 5(a). The relatively lower biotin-binding activity of E. coli cellswith a low concentration of biotin in comparison with magneticbeads could be explained by the lower mobility of E. coli in the reac-tion step (by slow mixing) than the magnetic beads, which resultsfrom (1) the relatively lower density of E. coli compared to magneticbeads and (2) the asymmetric structure of E. coli cells. Therefore, thepossibility of the surface-bound streptavidin meeting the biotin inthe solution is expected to be comparatively lower for E. coli cells

than the magnetic beads.

Recently, we presented that the outer membrane of E. coli canbe separated and layered on a microplate while conserving theorientation of outer membrane and its hydrophobic interactions

ith pST001 and the intact E. coli were analyzed after treatment with biotinylated

310 M. Park et al. / Enzyme and Microbial Technology 48 (2011) 307–311

Fig. 5. Biotin-binding activity of E. coli cell with autodisplayed streptavidin com-pared to streptavidin-coated magnetic beads. (a) Biotin-binding activity of magneticbeads with different amounts of immobilized streptavidin (n = 3). The maximumbiotin-binding activity was obtained when the concentration of the streptavidinsolution was higher than 100 ng/ml. (b) The biotin-binding activity of E. coli withautodisplayed streptavidin compared with the magnetic beads. The magnetic beadsa

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Fig. 6. Biotin-binding activity of the outer membrane layer of E. coli with autodis-

target enzyme labeling: identification of new human cathepsin G inhibitors.Anal Biochem 2005;46:258–67.

[6] Jose J. Autodisplay: efficient bacterial surface display of recombinant proteins.

nd E. coli reacted with the same concentration of biotinylated HRP (n = 3).

17,35]. In this work, outer membranes with autodisplayed strep-avidin were prepared as previously described [17,35]. The outer

embranes were layered on a 96-well microplate by incubatinghe separated outer membrane solution from cells at an OD578f 1.0 for 3 hat room temperature. The biotin-binding activity ofhe outer membrane layer with autodisplayed streptavidin wasstimated by treatment of biotinylated HRP followed by a chro-ogenic reaction with TMB. As shown in Fig. 6, the outer membrane

ayer with autodisplayed streptavidin showed a dynamic range ofiotin-binding activity, from 1 ng/ml to 300 ng/ml. This range isomparable to that of the E. coli cell-based assay, shown in Fig. 5(b).he outer membrane layer of intact E. coli showed a signal similar tohe baseline signal for the whole range of biotinylated HRP, whichndicates very low non-specific binding of proteins to the E. coliuter membrane.

These results show that the outer membrane layer with autodis-layed streptavidin has comparable biotin-binding activity totreptavidin on the autodisplayed E. coli cells and that the outer

embrane layer prepared on the microplate has potential applica-

ion in immunoassays.

played streptavidin. The biotin-binding activity was estimated through a reactionwith the chromogenic substrate (TMB) after treatment with biotinylated HRP. Tocompare the activity of streptavidin, the outer membrane layer of intact E. coli andphysically adsorbed biotinylated HRP were used as negative controls.

4. Conclusions

In this work, the autodisplay of streptavidin was presented.To confirm the presence of autodisplayed streptavidin, the outermembrane protein was analyzed by SDS-PAGE, and the numberof streptavidin molecules on a single E. coli cell was estimated tobe 1.6 × 105 molecules/cell by densitometric analysis. The biotin-binding activity of the autodisplayed streptavidin was estimatedafter treatment with fluorescently labeled biotin by fluorescencemicroscopy. From the FACS analysis, the efficiency of the autodis-play process was evaluated to be more than 95%. The biotin-bindingactivity of the E. coli with autodisplayed streptavidin was comparedwith similarly sized magnetic beads with covalently immobilizedstreptavidin. The outer membrane with autodisplayed streptavidinwas isolated and layered on a 96-well microplate, and the biotin-binding activity was estimated. Considering previously reportedapplications based on the capacity of E. coli cells to autodisplay pro-teins on its outer membrane layer, the autodisplayed streptavidinis expected to be potentially applicable to various immunoassayformats.

Acknowledgements

This research was supported by the National Research Founda-tion of Korea (NRF) funded by the Ministry of Education, Scienceand Technology (2009-0082188, 2009-0073809, 2009-008-1529,2009-62890, F01-2009-10124 and R15-2004-024-00000-0) and bythe “Happy Tech” program (2010-0020772 and 2010-0020767).

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