title: A novel screening system based on VanX-mediated autolysis-Application to Gaussia luciferase.
abstract: We report a novel bacterial screening protocol based on co-expressing the target protein with VanX, an enzyme which mediates Escherichia coli's autolysis and the release of the target protein into the culture medium, thereby facilitating activity measurement and screening from crude medium. This protocol as assessed with 19 Gaussia luciferase (GLuc) expressing colonies, was able to detect bioluminescence wavelength shift as small as 1.5 nm. We demonstrate the performance and versatility of this protocol by applying it to a semi-rational search for GLuc variants with red-shifted bioluminescence. Six GLuc's sites, F113, I114, W143, L144, A149, and F151, were randomly mutated, and for each site, 50 colonies were cultivated in 3 mL samples, from which bioluminescence was measured without purification. We identified two GLuc single mutation red-shifted variants: W143V and L144A. Their red shifted bioluminescence and biophysical/biochemical properties were confirmed using HPLC purified variants. Biotechnol. Bioeng. 2016;113: 1413-1420. © 2015 Wiley Periodicals, Inc.
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo, 184-8588, Japan.
authors.forename: Nan
authors.initials: N
authors.lastname: Wu
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo, 184-8588, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kamioka
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo, 184-8588, Japan. ykuroda@cc.tuat.ac.jp.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase
keywords: bacterial lysis; bioluminescence; co-expression; semi-random mutagenesis