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 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 foci 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.
Domingo
G J
GJ
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK.
Orru'
S
S
Perham
R N
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D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D049452:Green Fluorescent Proteins; D007114:Immunization; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D010961:Plasmodium / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013499:Surface Properties
D000939:Epitopes; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)