title: Content of intrinsic disorder influences the outcome of cell-free protein synthesis.
abstract: Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.
authors.affiliation: Research Center for Environmental Genomics, Kobe University, Nada 657-8501, Japan.|RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Tokmakov
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kurotani
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Mariko
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Terazawa
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: Department of Biochemistry, Saint-Petersburg State University, St. Petersburg 199034, Russia.
authors.forename: Vasily
authors.initials: V
authors.lastname: Stefanov
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D002474:Cell-Free System; D057927:Hydrophobic and Hydrophilic Interactions; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D054875:Ubiquitination
chemical_list: D011506:Proteins; D011994:Recombinant Proteins