Henri R. Nordlund

Genetically engineered avidins and streptavidins

Abstract.Chicken avidin and bacterial streptavidin, (strept)avidin, are proteins widely utilized in a number of applications in life science, ranging from purification and labeling techniques to diagnostics, and from targeted drug delivery to nanotechnology. (Strept)avidin-biotin technology relies on the extremely tight and specific affinity between (strept)avidin and biotin (dissociation constant, Kd≈10−14–10−16 M). (Strept)avidins are also exceptionally stable proteins. To study their ligand binding and stability characteristics, the two proteins have been extensively modified both chemically and genetically. There are excellent accounts of this technology and chemically modified (strept)avidins, but no comprehensive reviews exist concerning genetically engineered (strept)avidins. To fill this gap, we here go through the genetically engineered (strept)avidins, summarizing how these constructs were designed and how they have improved our understanding of the structural and functional characteristics of these proteins, and the benefits they have provided for (strept)avidin-biotin technology.

Biochemical characterization of CA IX: one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys119-Cys299) and a unique N-linked glycosylation site (Asn309) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr78) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Rational Design of an Active Avidin Monomer

Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 → 2 interface was mutated to lysine and asparagine 54 in the 1 → 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K d ∼10−15 m of the wild-type tetramer to K d ∼10−7 m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

Novel avidin-like protein from a root nodule symbiotic bacterium, Bradyrhizobium japonicum.

Bradyrhizobium japonicum is an important nitrogenfixing symbiotic bacterium, which can form root nodules on soybeans. These bacteria have a gene encoding a putative avidin- and streptavidin-like protein, which bears an amino acid sequence identity of only about 30% over the core regions with both of them. We produced this protein in Escherichia coli both as the full-length wild type and as a C-terminally truncated core form and showed that it is indeed a high affinity biotin-binding protein that resembles (strept)avidin structurally and functionally. Because of the considerable dissimilarity in the amino acid sequence, however, it is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin did not show significant cross-reactivity with it. Therefore this new avidin, named bradavidin, facilitates medical treatments such as targeted drug delivery, gene therapy, and imaging by offering an alternative tool for use if (strept)avidin cannot be used, because of a deleterious patient immune response for example. In addition to its medical value, bradavidin can be used both in other applications of avidin-biotin technology and as a source of new ideas when creating engineered (strept)avidin forms by changing or combining the desired parts, interface patterns, or specific residues within the avidin protein family. Moreover, the unexpected discovery of bradavidin indicates that the group of new and undiscovered bacterial avidin-like proteins may be both more diverse and more common than hitherto thought.

Chicken avidin-related proteins show altered biotin-binding and physico-chemical properties as compared with avidin

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 Lys(111)-->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.

Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins

The chicken avidin gene family consists of avidin and seven separate avidin-related genes (AVRs) 1–7. Avidin protein is a widely used biochemical tool, whereas the other family members have only recently been produced as recombinant proteins and characterized. In our previous study, AVR4 was found to be the most stable biotin binding protein thus far characterized (Tm = 106.4 °C). In this study, we studied further the biotin-binding properties of AVR4. A decrease in the energy barrier between the biotin-bound and unbound state of AVR4 was observed when compared with that of avidin. The high resolution structure of AVR4 facilitated comparison of the structural details of avidin and AVR4. In the present study, we used the information obtained from these comparative studies to transfer the stability and functional properties of AVR4 to avidin. A chimeric avidin protein, ChiAVD, containing a 21-amino acid segment of AVR4 was found to be significantly more stable (Tm = 96.5 °C) than native avidin (Tm = 83.5 °C), and its biotin-binding properties resembled those of AVR4. Optimization of a crucial subunit interface of avidin by an AVR4-inspired point mutation, I117Y, significantly increased the thermostability of the avidin mutant (Tm = 97.5 °C) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (Tm = 111.1 °C). This study provides an example of rational protein engineering in which another member of the protein family has been utilized as a source in the optimization of selected properties.

Chicken Avidin-related Protein 4/5 Shows Superior Thermal Stability when Compared with Avidin while Retaining High Affinity to Biotin

The protein chicken avidin is a commonly used tool in various applications. The avidin gene belongs to a gene family that also includes seven other members known as the avidin-related genes (AVR). We report here on the extremely high thermal stability and functional characteristics of avidin-related protein AVR4/5, a member of the avidin protein family. The thermal stability characteristics of AVR4/5 were examined using a differential scanning calorimeter, microparticle analysis, and a microplate assay. Its biotin-binding properties were studied using an isothermal calorimeter and IAsys optical biosensor. According to these analyses, in the absence of biotin AVR4/5 is clearly more stable (Tm = 107.4 ± 0.3 °C) than avidin (Tm = 83.5 ± 0.1 °C) or bacterial streptavidin (Tm = 75.5 °C). AVR4/5 also exhibits a high affinity for biotin (Kd ≈ 3.6 × 10-14 m) comparable to that of avidin and streptavidin (Kd ≈ 10-15 m). Molecular modeling and site-directed mutagenesis were used to study the molecular details behind the observed high thermostability. The results indicate that AVR4/5 and its mutants have high potential as new improved tools for applications where exceptionally high stability and tight biotin binding are needed.

Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding

In order to turn the subunit association and biotin binding of avidin into pH‐sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1–3 interface, and in combination with a histidine conversion in the 1–2 interface (Trp110). The single replacements Met96His and Val115His in the 1–3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH‐dependent. The histidine replacement in the 1–2 interface affected the biotin‐binding properties of the mutants, in particular reversibility of binding and protein–ligand complex formation were pH‐sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin–biotin technology.

Characterization of poultry egg-white avidins and their potential as a tool in pretargeting cancer treatment.

Chicken avidin and bacterial streptavidin are proteins used in a wide variety of applications in the life sciences due to their strong affinity for biotin. A new and promising use for them is in medical pretargeting cancer treatments. However, their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in these applications. To search for potentially beneficial new candidates, we screened egg white from four different poultry species for avidin. Avidin proteins, isolated from the duck, goose, ostrich and turkey, showed a similar tetrameric structure, similar glycosylation and stability against both temperature and proteolytic activity of proteinase K as chicken avidin. Biotin-binding properties of these avidins, measured using IAsys optical biosensor, were similar to those found in avidin from the chicken. Three of these novel avidins, however, showed different immunological cross-reactivities when compared with chicken avidin. The patient sera responses to duck, goose and ostrich avidins were also lower than those observed for chicken and turkey avidins. Our findings suggest that the use of these proteins offers advantages over chicken avidin and bacterial streptavidin in pretargeting applications.