APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY In vitro selection of scFv and its production: an application of mRNA display and wheat embryo cell-free and E. coli cell production system Tatsuro Shibui & Teruaki Kobayashi & Keiichiro Kanatani & Hirohisa Koga & Satoru Misawa & Tetsu Isomura & Tooru Sasaki Received: 6 February 2009 / Revised: 8 April 2009 / Accepted: 12 April 2009 / Published online: 7 May 2009 # Springer-Verlag 2009 Abstract Synthetic DNA libraries encoding human anti- body V L and V H fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the number of molecules used in mRNA display, the size of the library displayed was calculated to be >10 10 . To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established. Keywords mRNA display . Antibody . scFv . Wheat embryo cell-free . E. coli production Introduction Monoclonal antibodies (mAbs) are useful for biological research and diagnoses and have also been developed as drugs (Fogelman et al. 2008; Hall and Cameron 2008; Onal et al. 2008). Hybridoma technology (Bussard and Pages 1978; Geckeler et al. 1978; Shulman et al. 1978) has been used to produce mAbs for about 30 years. The process involves immunizing animals and preparing B cells to make hybridoma cells. In the past 15 years, phage display (Smith 1985; Smith and Scott 1993), which can display human proteins, has provided an alternative for the production of mAbs, mainly for therapeutic uses (Ranganathan 2008). Since the process does not require immunization and the making of hybridoma cells, antibodies can be obtained in a shorter period of time, and moreover, by using human antibody genes, human antibodies can easily be obtained. Recently, with advances in in vitro protein synthesis, in vitro display systems, i.e., ribosome display (Hanes and Pluckthun 1997) and mRNA display (Miyamoto-Sato et al. 2000; Nemoto et al. 1997; Shibui et al. 2008; Shiratori et al. 2008), have been developed and used for engineering proteins (Fukuda et al. 2006; Lipovsek and Pluckthun 2004). The Appl Microbiol Biotechnol (2009) 84:725–732 DOI 10.1007/s00253-009-2010-z T. Shibui (*) : T. Kobayashi : K. Kanatani : H. Koga : T. Isomura MOLECUENCE Corporation, Mitsubishi, Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan e-mail: 1807766@cc.m-kagaku.co.jp S. Misawa Mitsubishi Chemical Research Center, Mitsubishi, Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan T. Sasaki ZOEGENE Corporation, Mitsubishi, Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa 227-8502, Japan