Enzyme and Microbial Technology 66 (2014) 42–47 Contents lists available at ScienceDirect Enzyme and Microbial Technology jo ur nal ho me pa ge: www.elsevier.com/locate/emt Ultrasonic isolation of the outer membrane of Escherichia coli with autodisplayed Z-domains Ji-Hong Bong a , Gu Yoo a , Min Park b , Min-Jung Kang b , Joachim Jose c , Jae-Chul Pyun a,∗ a Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul 120-742, Republic of Korea b Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea c Muenster University, Muenster, Germany a r t i c l e i n f o Article history: Received 19 May 2014 Received in revised form 8 July 2014 Accepted 14 August 2014 Available online 23 August 2014 Keywords: E. coli Autodisplay Outer membrane Isolation Ultrasonication a b s t r a c t The outer membrane of Escherichia coli was previously isolated as a liposome-like outer membrane par- ticle using an enzymatic treatment for lysozymes; for immunoassays, the particles were subsequently layered on solid supports via hydrophobic interactions. This work presents an enzyme-free isolation method for the E. coli outer membrane with autodisplayed Z-domains using ultrasonication. First, the properties of the outer membrane particle, such as the particle size, zeta potential, and total protein, were compared with the properties of particles obtained using the previous preparation methods. Compared with the conventional isolation method using an enzyme treatment, the ultrasonic method exhibited a higher efficiency at isolating the outer membrane and less contamination by cytosolic proteins. The iso- lated outer membrane particles were layered on a gold surface, and the roughness and thickness of the layered outer membrane layers were subsequently analyzed using AFM analysis. Finally, the antibody- binding activity of two outer membrane layers with autodisplayed Z-domains created from particles that were isolated using the enzymatic and ultrasonic isolation methods was measured using fluorescein- labeled antibody as a model analyte, and the activity of the outer membrane layer that was isolated from the ultrasonic method was estimated to be more than 20% higher than that from the conventional enzymatic method. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Autodisplay technology has been used to express target pro- teins, which were previously expressed on the outer membrane of Escherichia coli [1,2]. The autotransporter domain of a protein called adhesin involved in diffuse adherence from E. coli (AIDA- I) for the outer membrane translocation of a target protein in combination with the signal peptide of the cholera toxin-subunit (CTB) and an artificial promoter [3]. Recently, we reported the autodisplay of the Z-domain, which had a specific binding activ- ity for the F c region of antibodies (IgG) [4,5]. The antibodies that bound to the Z-domains always had an active orientation that could bind antigens because the F ab region was exposed to antigens; thus, the sensitivity of immunoassays with orientation-controlled antibodies was reported to be greatly improved compared with that of the immunoassays that used randomly oriented antibodies [6]. The entire E. coli cell with autodisplayed Z-domains could be ∗ Corresponding author. Tel.: +82 2 2293 5509; fax: +82 2 312 5375. E-mail address: jcpyun@yonsei.ac.kr (J.-C. Pyun). used for immunoassays with various assay configurations, such as the sandwich-format assay on a microplate [7–9] and FACS-based assays [10–12]. Another configuration of immunoassays was also reported where only the outer membrane of E. coli was isolated, and the membrane was then layered on solid supports for immunoas- says, such as those on microplates [4], the metal surface of SPR biosensors [5] and microbeads [13]. The conventional method for isolating the outer membrane of E. coli used an enzymatic treatment of lysozyme to hydrolyze the pep- tidoglycan layer, after which the outer membrane was isolated as liposome-like outer membrane particles with a diameter of 100 nm [14,15]. Such outer membrane particles were known to have a hydrophilic surface and a hydrophobic core; via hydrophobic inter- actions between the hydrophobic core part of outer membrane particles and hydrophobic surfaces, the particles could form an outer membrane layer with a thickness of several nanometers on various hydrophobic surfaces of solid supports for immunoassays [4,5]. In this work, an enzyme-free method using ultrasonication to isolate the E. coli outer membrane was presented as shown in Fig. 1, and several advantages of the enzyme-free isolation by http://dx.doi.org/10.1016/j.enzmictec.2014.08.006 0141-0229/© 2014 Elsevier Inc. All rights reserved.