Binding and enrichment of Escherichia coli spheroplasts expressing inner membrane tethered scFv antibodies on surface immobilized antigens

Sang Taek Jung, Jun Jeong Ki, Brent L. Iverson, George Georgiou

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Anchored periplasmic expression (APEx) is a new method for the isolation of high affinity ligand-binding proteins from large combinatorial libraries (Harvey et al., 2004, Proc Natl Acad Sci USA 101(25): 9193-9198). In APEx, proteins are expressed as fusions to a membrane anchor that tethers them onto the periplasmic side of the Escherichia coli inner membrane. Conversion of the cells into spheroplasts and incubation with soluble fluorescently conjugated ligands results in the specific labeling of cells expressing ligand-binding proteins and their subsequent isolation by flow cytometry. Here we show that scFv antibody fragments expressed in the APEx format allow the binding of spheroplasts to immobilized ligands. ScFv antibodies specific for the cardiac glycoside digoxin or for the protective antigen (PA) of Bacillus anthracis as a negative control were expressed in E. coli as fusions to either N-terminal or C-terminal membrane anchoring domains. Only the C-terminally anchored fusions resulted in specific recognition and binding of spheroplasts onto TentaGel beads with immobilized antigen. Following three rounds of flow cytometric screening, spheroplasts expressing anti-digoxin scFvs were enriched 950-fold from a large excess (1,000x) of spheroplasts expressing anti-PA antibodies. These results indicate that the APEx technology may be employed for the screening of libraries based on binding to insoluble antigens possibly including antigens on cell surfaces.

Original languageEnglish
Pages (from-to)39-47
Number of pages9
JournalBiotechnology and Bioengineering
Volume98
Issue number1
DOIs
Publication statusPublished - 2007 Sep 1
Externally publishedYes

Fingerprint

Spheroplasts
Single-Chain Antibodies
Surface Antigens
Antigens
Antibodies
Escherichia coli
Ligands
Membranes
Fusion reactions
Digoxin
Carrier Proteins
Screening
Cardiac Glycosides
Immunoglobulin Fragments
Flow cytometry
Glycosides
Anchors
Labeling
Bacilli
Cells

Keywords

  • Anchored periplasmic expression
  • Enrichment
  • Immobilized antigens
  • scFv
  • Spheroplasts

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Binding and enrichment of Escherichia coli spheroplasts expressing inner membrane tethered scFv antibodies on surface immobilized antigens. / Jung, Sang Taek; Ki, Jun Jeong; Iverson, Brent L.; Georgiou, George.

In: Biotechnology and Bioengineering, Vol. 98, No. 1, 01.09.2007, p. 39-47.

Research output: Contribution to journalArticle

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