Construction of a dual chain pseudotetrameric chicken avidin by combining two circularly permuted avidins.
Two distinct circularly permuted forms of chicken avidin were designed with the aim of constructing a fusion avidin containing two biotin-binding sites in one polypeptide. The old N and C termini of wild-type avidin were connected to each other via a glycine/serine-rich linker, and the new termini were introduced into two different loops. This enabled the creation of the desired fusion construct using a short linker peptide between the two different circularly permuted subunits. The circularly permuted avidins (circularly permuted avidin 5 --> 4 and circularly permuted avidin 6 --> 5) and their fusion, pseudotetrameric dual chain avidin, were biologically active, i.e. showed biotin binding, and also displayed structural characteristics similar to those of wild-type avidin. Dual chain avidin facilitates the development of dual affinity avidins by allowing adjustment of the ligand-binding properties in half of the binding sites independent of the other half. In addition, the subunit fusion strategy described in this study can be used, where applicable, to modify oligomeric proteins in general.
Nordlund
Henri R
HR
NanoScience Center (NSC), Department of Biological and Environmental Science, P. O. Box 35, FIN-40014 University of Jyväskylä, Finland.
Laitinen
Olli H
OH
Hytönen
Vesa P
VP
Uotila
Sanna T H
ST
Porkka
Eevaleena
E
Kulomaa
Markku S
MS
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D002645:Chickens; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D012694:Serine / Q000737:chemistry
D010455:Peptides; D001360:Avidin; D012694:Serine; D001710:Biotin; D019286:Endopeptidase K; D005998:Glycine