Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin—biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys111→isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin—biotin technological applications.
- avidin–biotin technology
- biotin-binding protein
- molecular evolution
- molecular modelling
- structure–function relationship
↵1 Present address: A. I. Virtanen Institute, Department of Molecular Medicine, University of Kuopio, P.O. Box 1627, Kuopio, Finland.
↵2 Present address: National Public Health Institute, Department of Molecular Medicine, University of Helsinki, P.O. Box 104, FIN-00251 Helsinki, Finland.
Abbreviations used: AVR, avidin-related gene; AVD, avidin gene; AVR, avidin-related protein; 3-D, three-dimensional; RT-PCR, reverse-transcription PCR; Endo Hf, recombinant endoglycosidase H fused (f) to maltose-binding protein; PNGase F, peptide:N-glycosidase F; IgG—AP, IgG—alkaline phosphatase conjugate; L3 (etc.), loops designated by their numeral orders; β3 (etc.), β-sheets designated by their numeral orders; Kd, dissociation constant; kdiss, dissociation rate constant; kass, association rate constant; CPK, Corey—Pauling—Koltun.
- The Biochemical Society, London ©2002