Premium
A novel screening system based on VanX‐mediated autolysis—Application to Gaussia luciferase
Author(s) -
Wu Nan,
Kamioka Tetsuya,
Kuroda Yutaka
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25910
Subject(s) - bioluminescence , luciferase , autolysis (biology) , biology , escherichia coli , biochemistry , microbiology and biotechnology , enzyme , gene , transfection
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.