title: 'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.
abstract: A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Knowledge Hub Aichi, Aichi Science and Technology Foundation, Yakusa-cho, Toyota 470-0356, Japan teruyo.ojima@gmail.com hnakano@agr.nagoya-u.ac.jp.
authors.forename: Teruyo
authors.initials: T
authors.lastname: Ojima-Kato
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Kansuke
authors.initials: K
authors.lastname: Fukui
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yamamoto
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Dai
authors.initials: D
authors.lastname: Hashimura
authors.affiliation: Advanced Scientific Technology and Management Research Institute of Kyoto, Chudoji Minamimachi, Shimogyo-ku, Kyoto 600-8813, Japan.
authors.forename: Shiro
authors.initials: S
authors.lastname: Miyake
authors.affiliation: Advanced Scientific Technology and Management Research Institute of Kyoto, Chudoji Minamimachi, Shimogyo-ku, Kyoto 600-8813, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hirakawa
authors.affiliation: Advanced Scientific Technology and Management Research Institute of Kyoto, Chudoji Minamimachi, Shimogyo-ku, Kyoto 600-8813, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Yamasaki
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan teruyo.ojima@gmail.com hnakano@agr.nagoya-u.ac.jp.
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016350:Leucine Zippers / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins
keywords: Escherichia coli; cell-free protein synthesis; expression system; fragment of antigen binding; leucine zipper