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

The acyltransferase components (E2) from the family of 2-oxo acid dehydroge nase 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 o f Bacillus stearothermophilus assemble to form a pentagonal dodecahedral sc affold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is po ssible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, an d a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodie s in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of th e 60 copies of the E2 chain folded into its active form, as judged by its f luorescence and detection in localized foci in Escherichia coli cells in vi vo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be ac hieved by reversible denaturation and reassembly of mixtures of appropriate ly modified E2 chains. This new methodology offers several important advant ages over other current display technologies, not least in the size of inse rt that can be accommodated and the multiplicity of display that can be ach ieved. (C) 2001 Academic Press.