Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex

Gonzalo J. Domingo, Stefania Orru', Richard N. Perham

Research output: Contribution to journalArticle

Abstract

The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently. Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, and a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized loci in Escherichia coli cells in vivo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains. This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved.

Original languageEnglish
Pages (from-to)259-267
Number of pages9
JournalJournal of Molecular Biology
Volume305
Issue number2
DOIs
Publication statusPublished - Jan 12 2001

Keywords

  • Macromolecular scaffolds
  • Multienzyme complex
  • Peptide epitopes
  • Phage display
  • Pyruvate dehydrogenase

ASJC Scopus subject areas

  • Virology

Fingerprint Dive into the research topics of 'Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex'. Together they form a unique fingerprint.

  • Cite this