title: PURE mRNA display for in vitro selection of single-chain antibodies.
abstract: mRNA display is a method to form a covalent linkage between a cell-free synthesized protein (phenotype) and its encoding mRNA (genotype) through puromycin for in vitro selection of proteins. Although a wheat germ cell-free translation system has been previously used in our mRNA display system, a protein synthesis using recombinant elements (PURE) system is a more attractive approach because it contains no endogenous nucleases and proteases and is optimized for folding of antibodies with disulphide bonds. However, when we used the PURE system for mRNA display of single-chain Fv (scFv) antibodies, the formation efficiency of the mRNA-protein conjugates was quite low. To establish an efficient platform for the PURE mRNA display of scFv, we performed affinity selection of a library of scFv antibodies with a C-terminal random sequence and obtained C-terminal sequences that increased the formation of mRNA-protein conjugates. We also identified unexpected common substitution mutations around the start codon of scFv antibodies, which were inferred to destabilize the mRNA secondary structure. This destabilization causes an increase in protein expression and the efficiency of the formation of mRNA-protein conjugates. We believe these improvements should make the PURE mRNA display more efficient for selecting antibodies for diagnostic and therapeutic applications.
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Nagumo
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Horisawa
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan doi@bio.keio.ac.jp.
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
mesh_terms: D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics; D014908:Triticum / Q000737:chemistry / Q000378:metabolism
chemical_list: D057127:Single-Chain Antibodies
keywords: PURE system; antibody engineering; cell-free translation; puromycin; ribosome stalling