title: Solid-phase translation and RNA-protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA.
abstract: A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA-protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA-protein fusion is ligated to the 3' ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA-protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of 'RNA chip-to-protein chip' using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution.
authors.affiliation: Rational Evolutionary Design of Advanced Biomolecules, Saitama Small Enterprise Promotion Corporation SKIP city, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, Japan. biyanijp@yahoo.co.in
authors.forename: Manish
authors.initials: M
authors.lastname: Biyani
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D019020:Directed Molecular Evolution; D015723:Gene Library; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010641:Phenotype; D040081:Protein Array Analysis; D014176:Protein Biosynthesis; D017510:Protein Folding; D040901:Proteomics; D011691:Puromycin / Q000737:chemistry; D011786:Quality Control; D012333:RNA, Messenger / Q000737:chemistry*; D013698:Templates, Genetic
chemical_list: D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D011691:Puromycin; D000449:Aldehyde Reductase