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

Hyeok Jin Ko, Eunhye Park, Joseph Song, Taek Ho Yang, Hee Jong Lee, Kyoung Heon Kim, In-Geol Choi

Research output: Contribution to journalArticle

10 Citations (Scopus)

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 6.1×104 molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli.

Original languageEnglish
Pages (from-to)3051-3058
Number of pages8
JournalApplied and Environmental Microbiology
Volume78
Issue number9
DOIs
Publication statusPublished - 2012 May 1

Fingerprint

genetic vectors
Genetic Vectors
secretion
Ligation
enzyme
Escherichia coli
protein
Enzymes
enzymes
Proteins
proteins
cells
Tobacco etch virus
Type V Secretion Systems
cloning
Organism Cloning
Membrane Proteins
tobacco
cleavage
proteinases

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

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. / Ko, Hyeok Jin; Park, Eunhye; Song, Joseph; Yang, Taek Ho; Lee, Hee Jong; Kim, Kyoung Heon; Choi, In-Geol.

In: Applied and Environmental Microbiology, Vol. 78, No. 9, 01.05.2012, p. 3051-3058.

Research output: Contribution to journalArticle

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