Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications

Matthias Peipp, Domenica Saul, Karin Barbin, Joerg Bruenke, Susan J. Zunino, Michael Niederweis, Georg H. Fey

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 μg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcγRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

Original languageEnglish (US)
Pages (from-to)265-280
Number of pages16
JournalJournal of Immunological Methods
Volume285
Issue number2
DOIs
StatePublished - Feb 15 2004
Externally publishedYes

Keywords

  • Antibody engineering
  • CD antigen
  • Fc receptor
  • GFP
  • scFv

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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