title: Functional cell surface display and controlled secretion of diverse Agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL.
abstract: Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 10(4) molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.
authors.affiliation: Computational and Synthetic Biology Laboratory, College of Life Sciences and Biotechnology, Korea University, Seoul, South Korea.
authors.forename: Hyeok-Jin
authors.initials: HJ
authors.lastname: Ko
authors.forename: Eunhye
authors.initials: E
authors.lastname: Park
authors.forename: Joseph
authors.initials: J
authors.lastname: Song
authors.forename: Taek Ho
authors.initials: TH
authors.lastname: Yang
authors.forename: Hee Jong
authors.initials: HJ
authors.lastname: Lee
authors.forename: Kyoung Heon
authors.initials: KH
authors.lastname: Kim
authors.forename: In-Geol
authors.initials: IG
authors.lastname: Choi
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D029968:Escherichia coli Proteins; D006026:Glycoside Hydrolases; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C573499:YfaL protein, E coli; C019624:agarase