Patent Application
20100022401
The present invention relates to novel avidin-like proteins and a method for producing thereof, genes encoding the proteins, and methods for using the proteins and the genes. Specifically, it relates to a native and a truncated high affinity biotin-binding protein originated from Bradyrhizobium japonicum which proteins resemble (strept)avidin structurally and functionally.
Several avidin proteins have been found in bird, reptile and amphibian species (Hertz and Sebrell, 1942; Jones, 1962; Korpela et al., 1981). Those of the bird avidins that have been characterised are relatively similar, though displaying some differences in stability and immunological cross-reactivity when compared to those of chicken avidin (Hytönen et al., 2003; Korpela et al., 1981). In the chicken the avidin gene forms a gene family together with the avidin-related genes (AVR) (Ahlroth et al., 2000). These AVR proteins have recently been produced as recombinant proteins. Their characterisation and comparison with each other and with avidin revealed some differences in the properties of stability, glycosylation and biotin-binding, although the primary amino acid sequences are rather well conserved (Hytönen et al., 2004b; Laitinen et al., 2002). Several Streptomyces strains were studied four decades ago, and the bacterial analog for avidin, streptavidin, was found in a strain, which was given the name S. avidinii (Chaiet and Wolf, 1964). Inspired by these studies, other Streptomycetes have since been studied. So far two new streptavidins have been found in S. venezuelae and have been named accordingly streptavidin v1 and v2. These new forms were found to be almost identical with streptavidin, displaying only one (v1) or five (v2) amino acid substitutions in the core region, and with no observed significance for either the structure or the function of these proteins (Bayer et al., 1995).
Biotin is an essential cofactor in many vital biochemical reactions (Samols et al., 1988; Wood and Barden, 1977). Therefore it is understandable that (strept)avidin can work as a broad-range antimicrobial agent by forming a biotin free zone or protective barrier around an organism or, for example, an egg possessing it (Green, 1975). The biological role of bradavidin could also be protective as it proved to be a high affinity biotin binding protein. If the B. japonicum-containing root nodules on soybeans are found to express, possibly upon injury or infection, and contain or secrete at least a small amount of bradavidin, among other defence proteins and compounds, the plant could be resistant towards many invaders. These could include harmful soil microbes, insects and also higher animals. Experiments on transgenic corn have shown that the expression of avidin in the plant has an enormous impact on the majority of insect pests, particularly at certain developmental stage of the larvae (Kramer et al., 2000; Morgan et al., 1993).
Medical applications of avidin-biotin technology (Wilchek and Bayer, 1990; Wilchek and Bayer, 1999) include, for example, gene therapy (Lehtolainen et al., 2003; Wojda et al., 1999), imaging (Rosebrough, 1996) and targeted drug delivery (Lehtolainen et al., 2002; Räty et al., 2004). In traditional 1-step radioimmunotherapy (RIT) a therapeutic radioactive material is directly linked to a tumor-specific antibody (Beaumier et al., 1991; Klein et al., 1989; Knox et al., 1992). In order to improve the low target/non-target ratio, which is a drawback with this methodology, several improved protocols for delivering tumor cell-targeted radiation, which usually include more steps, have been developed (Boerman et al., 2003). One of the most promising methods is the 3-step pretargeting radioimmunotherapy (PRIT), which includes the following steps: (i) a biotinylated antibody specific for the target tumor cells, (ii) chicken avidin (fast pharmacokinetic clearance) as a clearing agent to remove endogenous biotin and the excess free circulating biotinylated antibodies from the first step, followed by streptavidin (slow pharmacokinetic clearance) which is mainly responsible for avidinylation of the tumor cells, and (iii) biotinylated radioactive material, which binds tightly to the free binding sites of the tetravalent (strept)avidin molecules immobilised by the biotinylated antibodies (Grana et al., 2002; Paganelli et al., 1999; Paganelli et al., 1991).
In addition to chicken avidin and streptavidin, the existing, rather thoroughly characterised avidin protein pool for medical purposes includes poultry avidins, of which duck, goose and ostrich avidin (Hytönen et al., 2003) in particular have been shown in vitro to be potential alternatives for patients who have strong immunological response toward (strept)avidin owing to usually repeated treatments. Some of the AVR proteins (Laitinen et al., 2002) might also prove useable instead of or before (strept)avidin in sequential PRIT treatments, if they turn out to be immunologically different enough in vivo and show no significant crossreactivity with the antibodies elicited in the possible preceding steps.
Furthermore, differences in pharmacokinetics and other properties owing, for example, to varied glycosylation patterns and protein pI (Rosebrough and Hartley, 1996), can be exploited when selecting avidins for specific applications. However, as all these biotin-binding proteins are xenoproteins, they are likely to be antigenic, and therefore cannot be used effectively on successive occasions with the same patient. Therefore, an immediate need exists for new and dissimilar avidins, such as the characterised bradavidin and the others discussed in this report.
Bradyrhizobium japonicum is an important nitrogen-fixing 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. The inventors produced this protein in E. coli both as the full length wild-type (SEQ ID NO: 1) and as a C-terminally truncated core (SEQ ID NO:2) forms, and showed that it is indeed a high affinity biotin-binding protein which resembles (strept)avidin structurally and functionally.
Here the expression “resembles structurally and functionally” refers to a protein which can fold to form a 3D-structure spatially like that of (strept)avidin and which can act similarly, for example by containing the crucial amino acid residues for substrate binding. However, other parts of the amino acid sequence, or secondary structure, may be substantially different as well as the immunological properties.
Owing to the considerable dissimilarity in the amino acid sequence, however, the avidin-like protein of the invention is immunologically very different, and polyclonal rabbit and human antibodies toward (strept)avidin do 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, due to a deleterious patient immune response for example.
In addition to its medical value, bradavidin can both be used in other applications of avidin-biotin technology as well as a source of new ideas when creating engineered (strept)avidin forms by changing or combining 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.
Accordingly, the first aspect of the present invention is an isolated protein that is structurally and functionally avidin-like with improved properties compared to native avidin or streptavidin and avidin-related proteins, AVRs, and an amino acid sequence having 40% or less, preferably about 30% or less homology, with avidin or streptavidin and highly conserved fingerprint having the sequence:
Here the highly “conserved fingerprint” describes a systematic and logical arrangement of conserved amino acids on a sequence. This fingerprint has been assembled through studies on tertiary structures and simulated binding interactions of known AVR proteins and their ligands. This pattern fits onto avidin, streptavidin and bradavidin sequences, even though the homology between these three is not very high. The fundamental factor is the location of the key amino acid residues in 3D-space while other residues connected to the protein backbone facilitate the correct foundation to reach these positions. By posing these key residues into accurate secondary arrangement and by appropriately limiting the distance between the fixed positions, allowing only small fluctuation between these essential avidin characteristics, a search string to select sequences fulfilling the requirements can be designed. By searching through databases, for example DNA libraries, the proteins of interest can be selected using this probe. Most of the prospective search hits have a strong potential to be avidins.
Although avidin and streptavidin are structurally and functionally similar, their pharmacokinetic characteristics differ radically (Rosebrough, 1993; Rosebrough and Hartley, 1996; Schechter et al., 1990). Both glycosylation and high pI are thought to cause the rapid clearance of avidin from the blood. It has been found that glycosylation causes the avidin accumulation in the liver, and the high pI is responsible for the avidin accumulation in the kidneys (Yao et al., 1999). Streptavidin, which has a significantly longer plasma-half life when compared to avidin, is known to accumulate in the kidneys (Schechter et al., 1990), possibly via integrin-mediated cell adhesion dependent on an RGD-like domain (Alon et al., 1993). A streptavidin mutant, in which this RGD-like stretch was modified, showed markedly reduced cell adhesion (Murray et al., 2002). Several attempts have been made to modify (strept)avidin in order to change their in vivo accumulation, clearance and immunological properties. Chinol et al. were able to lower avidin accumulation in the kidneys and liver by attaching polyethylene glycol (PEG) groups to avidin (Chinol et al., 1998). Furthermore, these PEGylated avidins were found to be less immunogenic. Also epitope-modified recombinant streptavidins, carrying point mutations, with markedly improved immunological properties, have been generated (Meyer et al., 2001). In another study, deglycosylated and chemically neutralized avidin was found to be superior when compared to wt avidin in brain delivery (Kang and Pardridge, 1994). The better penetration was supposed to be due to the extended circulation time. Analogously, when galactose moieties were chemically attached to streptavidin, its blood clearance was accelerated (Rosebrough and Hartley, 1996). Yao et al. in turn made other peculiar observations. They demonstrated that avidin itself accumulated efficiently in lectin-expressing tumors, whereas streptavidin and chemically neutralized avidin did not exhibit this kind of behaviour (Yao et al., 1998).
The biotin-binding properties of bradavidin were shown to bear more resemblance to streptavidin than avidin. Bradavidin displayed the fastest dissociation rate, when radio-biotin was used in the analysis. Avidin showed clearly the slowest dissociation, whereas the value for streptavidin fell between these two. Moreover, when the ligand was a fluorescent biotin conjugate, avidin was clearly the fastest in dissociation, and streptavidin and bradavidin were nearly identical showing a very slow and small release in the assay. This is in line with a previous study (Pazy et al., 2002), in which streptavidin was proven to be better biotin conjugate binder than avidin. This property, also characteristic of bradavidin, is interesting and renders it a good tool in applications, since the biotin in use is usually a conjugate and thus good affinity is essential. The structural differences in the loop between β-strands 3 and 4 are thought to explain this divergent binding ability of avidin and streptavidin (Livnah et al., 1993; Pazy et al., 2002; Weber et al., 1989). In bradavidin this loop is extraordinary, since it probably contains a cysteine residue, which forms a disulfide bridge with the cysteine residue on the structurally neighbouring loop between β-strands 5 and 6. This interesting motif, potentially on top the entrance of the binding site, could have some effect on the binding parameters described above. It could also explain the fundamental reasons behind them, such as the divergent association rate, which are intended to be studied in greater detail together with the possible crystal structure of bradavidin.
These characteristics are here referred among “improved properties”. These properties are assessed comparing against those known for thoroughly studied chicken avidin (SEQ ID NO:6) and streptavidin (SEQ ID NO:7). Other examples of improved properties are better affinity towards biotin conjugate, faster biotin dissociation rate, useful immunological properties and beneficial protein/protein-, protein/DNA or protein/ligand interaction or a lack of it, compared to avidin and streptavidin. The table 1 in example 3 is informative presenting measured characteristics for avidin, streptavidin and bradavidin.
The existence of the bradavidin gene in the genome of a root nodule symbiotic bacterium, B. japonicum, may be just the first example of other genes producing functionally similar proteins in other plant-related bacteria. It is possible that further study of the root nodules of species other than the soy-bean will reveal a variety of such proteins. According to the 16S ribosomal RNA gene comparison, the strains producing the different streptavidins (S. avidinii and S. venezuelae) described so far share about 97% sequence identity, indicating close evolutionary relationship. On the contrary, B. japonicum is clearly not a close relative of these bacteria, since its 16S rRNA gene bears only about 74% and 77% sequence identity with those of S. avidinii and S. venezuelae, respectively. A straightforward assumption would be that some of the possible new avidins from symbiotic bacteria closely resemble bradavidin, although completely different forms might also be found.
Another aspect of the invention is a gene encoding an avidin-like protein according to SEQ ID NO:3 or 4. Yet another aspect of the invention is a recombinant vector comprising the any of said genes, a transformant obtained by introducing said recombinant vector to a host organism or a recombinant protein produced by the transformant.
In addition to sequence database queries based on whole sequence similarity, an effective string could be obtained from an extensive multiple sequence alignment of different avidins and related biotin-binding proteins (
Therefore, another aspect of the invention is a method for searching avidin-like proteins from databases comprising use of a search string. An example of such a search string is presented in example 5 and its use is illustrated in
The comparison of some potential avidin-like sequences, with those of the avidin and avidin-related sequences, shown in the multiple sequence alignment (
The loops connecting β-strands three and four in avid in and streptavidin (
Moreover, W70 and W97 form part of the hydrophobic cavity of the ligand-binding site in avidin (Livnah et al., 1993). The importance of the equivalent residues in streptavidin for biotin binding has also been experimentally shown by Chilkoti et al. (Chilkoti et al., 1995), who mutated these residues to ala nine and phenylalanine and observed a significant decrease in affinity in the case of the alanine mutants. However, in the case of the phenylalanine substitutions the decreases in the observed affinities were only mediocre. It is, therefore assumed, that the W97Y difference present in the putative Burk_pseudomallei avidin would not radically diminish the biotin affinity of this protein. In the present study it was found that bradavidin is a high-affinity biotin-binding protein, although it has F at position 70 and avidin has W the same position, which further supports the idea that conservation of the complementarity of the binding site and the ligand structure is essential for high affinity (Livnah et al., 1993; Weber et al., 1989).
The most radical differences in the multiple sequence alignment are found in the loop around W110, which plays a central role in ligand binding (Chilkoti et al., 1995; Laitinen, 1999 #32; Laitinen et al., 1999; Laitinen et al., 2003; Livnah et al., 1993; Weber et al., 1989). It could be speculated that the loop structures containing the proline residues in Rhiz and Brad2 sequences (
The invention will be further described with reference to the following figures and non-limiting examples 1-5.
The gene coding for bradavidin (DBJ AP005955.1) was amplified by PCR using B. japonicum genomic DNA as a template, and extended using SES-PCR (Majumder, 1992) to include attL recombination sites at both ends (Hartley et al., 2000). Two constructs were generated: the full length wild-type (138 amino acid residues, SEQ. ID NO:1) and a C-terminally truncated core form (118 amino acid residues, SEQ ID NO:2). Both constructs contained also their innate signal peptides (25 amino acid residues), which is represented together with the wild type protein (163 amino acid residues) in SEQ ID NO:5. These constructs were then transferred to pBVboostFG vector (Laitinen O. H. et al., manuscript) using the site-specific recombination-based Gateway method (invitrogen). The resulting expression vectors were confirmed to be as designed by DNA sequencing.
E. coli BL-21 (AI) cells (Invitrogen) were used for protein expression as described previously (Hytönen et al., 2004a). The recombinant proteins were isolated from bacterial cell extracts by one-step affinity chromatography on 2-iminobiotin agarose column (Hytönen et al., 2004a). Eluted proteins were analysed by SDS-PAGE and subsequent Coomassie staining of the gels. The proteins appeared to be pure and virtually homogenous, as only one band per lane of the expected size was observed on gels. Protein concentrations were determined using the calculated extinction coefficient 39 380 M-1 cm-1 for both bradavidins at 280 nm (Gill and von Hippel, 1989).
Pairwise sequence alignments were done using the Needle program from the EMBOSS (European Molecular Biology Open Software Suite) package and the ClustalW program was used to generate the multiple sequence alignment (Thompson et al., 1994). The theoretic biochemical properties were determined using the ProtParam program (Gill and von Hippel, 1989). The putative signal peptide cleavage site was determined by the SignalP 3.0 program (Bendtsen et al., 2004).
Pairwise sequence alignment for mature core regions of avidin and bradavidin revealed that 29.2% of the amino acids are identical and 39.2% similar, whereas with streptavidin these values are 30.2% and 41.7%, respectively. Interestingly, when avidin and streptavidin are compared equivalently the values obtained, 31.9% and 45.2%, are only slightly higher. Multiple sequence alignment of streptavidin, bradavidin and avidin (
Ligand binding properties were studied both by [3H]biotin assay and fluorescent biotin conjugate assay. The dissociation rate constant of [3H]biotin (Amersham) from the bradavidins and avidin was measured at various temperatures as described in detail previously (Klumb et al., 1998). According to the results, (Table I,
The purified proteins were analysed by gel filtration using a Shimadzu HPLC instrument equipped with a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech, Uppsala, Sweden) with 50 mM Na-carbonate buffer (pH 11) with 150 mM NaCl as the liquid phase. The column was calibrated using a marker mixture (thyroglobulin, IgG, ovalbumin, myoglobin, vitamin B-12; Bio-Rad Laboratories, Hercules, Calif., U.S.A) and bovine serum albumin (Roche Diagnostics, Mannheim, Germany) as molecular mass standards.
Gel filtration chromatography showed that bradavidin is a homogenous tetramer and both forms appeared as a single symmetrical and sharp peak on the chromatograms. SDS-PAGE stability analysis confirmed the tetrameric appearance. These quaternary structures showed comparable stability with those of avidin and streptavidin (Table I).
The thermal stability characteristics of the proteins were studied by a SDS-PAGE based method as previously described in detail by (Bayer et al., 1996).
The apparent molecular mass is indicated and followed by the theoretical mass in brackets. Transition temperature (Tr) indicates the temperature in which half of the protein is tetrameric and half monomeric in the absence (first value) and presence (second value) of biotin. In addition to the measured dissociation rate constant (kdiss), the release percentage of the fluorescent biotin conjugate in one hour in the presence of excess free biotin is indicated. Calculated isoelectrical point (pI) and the number of cysteine residues per monomer are also indicated.
85
85
aThese values were obtained from Bayer et al. (Bayer et al., 1996)
Serum samples from cancer patients exposed to avidin and streptavidin were used to compare the immunological properties of the avidins. The serum samples as well as the negative control sera from persons not exposed to (strept)avidin were obtained from the Division of Nuclear Medicine, European Institute of Medicine, Milan, Italy. The analysis was performed similarly as described previously (Hytönen et al., 2003): Immobilizer™ Amino-plates (Nalge Nunc Int.) were coated with the proteins under study (10 μg/ml) in 100 mM Na-phosphate pH 7.5, agitated for one hour at room temperature and blocked with PBS-T (PBS+Tween 20 0.05% v/v). The serum samples were diluted 1:100 in PBS-T and incubated in the wells for one hour at 37° C. After washing three times with PBS-T, polyvalent anti-human immunoglobulin alkaline phosphatase (AP) conjugate (Sigma) was used as a secondary antibody (dilution 1:6000; 1 h, 37° C.), followed by six washes with PBS-T. Finally, p-nitrophenyl phosphate (1 mg/ml, Sigma) was used as a substrate molecule, and a plate reader was used to measure the absorbance at 405 nm.
Immunological cross-reactivity of bradavidin with human and rabbit serum antibodies, elicited toward avidin and streptavidin, analysed by an ELISA assay is illustrated in
Proteins were further compared using polyclonal rabbit antibodies produced against avidin (University of Oulu, Finland) and streptavidin (Weissman Institute, Jerusalem, Israel). Proteins were first attached to Immobilizer™ Amino plates as described above and blocked with PBS-T. Antibodies were diluted 1:2000 to PBS-T and applied to the protein-coated plates (1 h, 37° C.). After washing with PBS-T, goat anti-rabbit IgG AP (Bio-Rad Laboratories) diluted 1:2000 in PBS-T was used as a secondary antibody (1 h, 37° C.), and the signal was measured as above.
When bradavidin was probed by polyclonal anti-(strept)avidin rabbit antibodies on western blots, only the positive (strept)avidin controls were detected after immunostaining. Preceding that, when the nitro-cellulose filter was stained with Ponceau S-dye, wild-type bradavidin was clearly visible at the expected location whereas the bradavidin core appeared to be virtually absent from the blot at this stage (data not shown). This behaviour may result from the rather low pI of the core form (Table I), as also suspected, for example, in the case of the acidic natural rubber latex allergen Hev b5 (Akasawa et al., 1996).
A multiple sequence alignment of many known avidin-like proteins (Chaiet and Wolf, 1964; Green, 1975; Laitinen et al., 2002) and some new candidates suggested biotin binding capability for the open reading frames from Xanthomonas campestris (GenBank AE012315.1), Rhizobium etli (GenBank U80928.4), Bradyrhizobium japonicum (another candidate in addition to bradavidin, DBJ AP005940.1), Burkholderia pseudomallei (EMB BX571965.1) and Burkholderia mallei (GenBank CP000010.1). The majority of the putative biotin-binding residues, according to the avidin (Livnah et al., 1993) and streptavidin (Weber et al., 1989) structures, were conserved albeit the overall sequence similarity was rather low (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20041396 | Oct 2004 | FI | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI2005/000467 | 10/28/2005 | WO | 00 | 10/15/2009 |
