title: Extraction of recombinant protein from Escherichia coli by using a novel cell autolysis activity of VanX.
abstract: Escherichia coli is a versatile, low-cost, and popular host for expressing recombinant proteins. However, extracting recombinant proteins from E. coli requires cell wall breakage, which is both time- and effort-consuming. Here we report a novel cell breakage method based on our recent finding that VanX, which is a d-Ala-d-Ala dipeptidase encoded in a vancomycin-resistant VanA gene cluster, exhibits a strong cell lysis activity when expressed in isolation in E. coli. In our strategy, we coexpress VanX with the target protein, causing cell autolysis and release of the cellular content into the culture medium. We demonstrated this strategy for two model proteins, a green fluorescent protein variant (GFPuv) and Gaussia luciferase, and optimized the autolysis conditions and coexpression vectors. The fluorescence activity of GFPuv collected from the medium was identical to that of GFPuv purified by conventional methods. Cell breakage by VanX-mediated autolysis is very simple to implement and will efficiently complement traditional methods.
authors.affiliation: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kamioka
authors.forename: Shihori
authors.initials: S
authors.lastname: Sohya
authors.forename: Nan
authors.initials: N
authors.lastname: Wu
authors.forename: Tei
authors.initials: T
authors.lastname: Maki
authors.forename: Tomoki
authors.initials: T
authors.lastname: Matsuda
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.forename: Haruki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kuroda
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001433:Bacteriolysis / Q000187:drug effects*; D003470:Culture Media; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D049452:Green Fluorescent Proteins; D008156:Luciferases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase
keywords: Bacterial cell wall; Cell lysis; Coexpression; GFP; Protein purification; VanX