Display of green fluorescent protein on Escherichia coli cell surface

Huidong Shi, Wei Wen Su

Research output: Contribution to journalArticle

61 Citations (Scopus)

Abstract

In this study, expression of green fluorescence protein (GFP) on the external surface of Escherichia coli was achieved by construction of a fusion protein between Lpp-OmpA hybrid and a GFP variant, GFPmut2. The GFP was fused in frame to the carboxyl-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. Western blot analysis of membrane fractions identified the Lpp-OmpA-GFP fusion protein with the expected size (43 kDa). Immunofluorescence microscopy, immunoelectron microscopy, protease and extracellular pH sensitivity assays further confirmed that GFP is anchored on the outer membrane. The GFP displayed on the E. coli outer surface retained its fluorescence and was not susceptible to the indigenous outer membrane protease OmpT even though there are two putative OmpT proteolytic sites present in GFP. Optimization of the expression conditions was conducted using fluorometry, eliminating cumbersome immuno-labeling procedures. Surface-displayed GFP could be used in a variety of applications including screening of polypeptide libraries, development of live vaccines, construction of whole cell allosteric biosensors, and signal transduction studies. (C) 2001 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)25-34
Number of pages10
JournalEnzyme and Microbial Technology
Volume28
Issue number1
DOIs
StatePublished - Jan 2 2001

Fingerprint

Green Fluorescent Proteins
Escherichia coli
Display devices
Fluorescence
Proteins
Fusion reactions
Membranes
Peptide Hydrolases
Microscopic examination
Fluorometry
Immunoelectron Microscopy
Signal transduction
Escherichia coli Proteins
Biosensing Techniques
Vaccines
Polypeptides
Fluorescence Microscopy
Biosensors
Signal Transduction
Labeling

Keywords

  • Escherichia coli
  • Green fluorescence protein
  • Protein display

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Display of green fluorescent protein on Escherichia coli cell surface. / Shi, Huidong; Wen Su, Wei.

In: Enzyme and Microbial Technology, Vol. 28, No. 1, 02.01.2001, p. 25-34.

Research output: Contribution to journalArticle

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