Juha A. E. Määttä

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.

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.

Chimeric avidin shows stability against harsh chemical conditions-biochemical analysis and 3D structure.

Avidin and its bacterial analog streptavidin have been widely used in applications in life sciences. Recently, we described a highly thermostable engineered avidin, called chimeric avidin, which is a hybrid of avidin and avidin‐related protein 4. Here, we report a protocol for pilot‐scale production in E. coli and the X‐ray structure of chimeric avidin. The ligand‐binding properties of chimeric avidin were explored with isothermal titration calorimetry. We found chimeric avidin to be more stable against various harsh organic solvents at elevated temperatures compared to avidin and streptavidin. The properties of chimeric avidin make it a potential tool for new applications in biotechnology. Biotechnol. Bioeng. 2011; 108:481–490.

Bradavidin II from Bradyrhizobium japonicum: a new avidin-like biotin-binding protein.

A gene encoding an avidin-like protein was discovered in the genome of B. japonicum. The gene was cloned to an expression vector and a protein, named bradavidin II, was produced in E. coli. Bradavidin II has an identity of 20-30% and a similarity of 30-40% with previously discovered bradavidin and other avidin-like proteins. It has biochemical characteristics close to those of avidin and streptavidin and binds biotin tightly. In contrast to other tetrameric avidin-like proteins studied to date, bradavidin II has no tryptophan analogous to the W110 in avidin (W120 in streptavidin), thought to be one of the most essential residues for tight biotin-binding. Homology modeling suggests that a proline residue may function analogously to tryptophan in this particular position. Structural elements of bradavidin II such as an interface residue pattern or biotin contact residues could be used as such or transferred to engineered avidin forms to improve or create new tools for biotechnological applications.

Avidin related protein 2 shows unique structural and functional features among the avidin protein family

BackgroundThe chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.ResultsIn the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1–3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4.ConclusionAltogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology.

Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis

BackgroundAvidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog.ResultsXenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin.ConclusionThe novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.

Rational Modification of Ligand‐Binding Preference of Avidin by Circular Permutation and Mutagenesis

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4→3) that lacks the loop between β‐strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4′‐hydroxyazobenzene‐2‐carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4→3 even further, we mutated asparagine 118 on the bottom of the ligand‐binding pocket to methionine, which simultaneously caused a significant drop in biotin‐binding affinity. The X‐ray structure of cpAvd4→ 3(N118M) allows an understanding of the effect of mutation to biotin‐binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion‐fold between wild‐type avidin and cpAvd4→3(N118M). To demonstrate the versatility of the cpAvd4→3 construct, we have shown that it is possible to link cpAvd4→3 and cpAvd5→4 to form the dual‐chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self‐organising nanoscale avidin building blocks.

Protein−Protein Interactions: Inhibition of Mammalian Carbonic Anhydrases I−XV by the Murine Inhibitor of Carbonic Anhydrase and Other Members of the Transferrin Family

The murine inhibitor of carbonic anhydrase (mICA), a member of the transferrin (TF) superfamily of proteins, together with human holo- and apoTF and lactoferrin (LF) were assessed as inhibitors of all catalytically active mammalian (h = human, m = murine) CA isoforms, from CA I to CA XV. mICA was a low nanomolar to subnanomolar inhibitor of hCAs I, II, III, VA, VB, VII and mCAs XV (K(I) of 0.7-44.0 nM) and inhibited the remaining isoforms with K(I) of 185.5-469 nM. hTF, apoTF, and hLF were inhibitors of most of these CAs but with reduced efficiency compared to mICA (K(I) of 18.9-453.8 nM). Biacore surface plasmon resonance and differential scanning calorimetry experiments were also used for obtaining more insights into the interaction between these proteins, which may be useful for drug design of protein-based CA inhibitors.

Talin-bound NPLY motif recruits integrin-signaling adapters to regulate cell spreading and mechanosensing

β3 integrin residue Y747 is required for cell spreading and paxillin adapter recruitment to substrate-bound integrins in response to substrate stiffness.

Switchavidin: Reversible Biotin–Avidin–Biotin Bridges with High Affinity and Specificity

Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.