FIELD OF THE INVENTION

The present invention relates to a method of generating and selecting novel avidin forms binding testosterone and other target molecules. The method is based on targeted randomization of selected loop regions in avidin and selection of proteins with novel ligand-binding characteristics by phage display methodology.

BACKGROUND OF THE INVENTION

Avidin is a tetrameric protein isolated from chicken egg white. It binds biotin with very hhiigghh aaffffiinniittyy ((KK _dd ~~ 1100 ^~"1155 MM)).. DDuuee ttoo ttiigghhtt aanndd ssppeeccific binding, avidin-biotin pair is widely involved in biotechnology (Laitinen et al., 2006).

Avidin is a eukaryotic protein and the efficient expression of active recombinant avidin in E. coli can be achieved by the use of bacterial signal peptide (Hytδnen et al., 2004). Avidins belong to calycin superfamily, which contains wide array of beta-barrel proteins. Many of the calysins bind small ligands. An example of previous protein engineering studies with calycins are modified proteins called anticalins, which bind, for example, fluorescein (Beste et al., 1999) and digoxigenin (Schlehuber et al. 2000). These novel anticalin proteins were engineered from lipocalin fold which is a beta-barrel calycin protein (US Patent No: 7,250,297). Although avidin, like lipocalins, is also beta-barrel protein, avidin and lipocalins are not equivalents in architecture and function. Lipocalins are mainly monomeric or dimeric proteins, (Flower, 1996) whereas avidin is a homotetramer (Green, 1975)

Avidin offers an exceptional platform for development of novel biotechnology tools. There is ongoing active development of avidin variants (Laitinen et al., 2006) and the structural properties of the protein and some of the protein variants are well known (Livnah et al., 1993; Eisenberg-Domovich et al., 2005; Hytδnen et al., 2005b). The avidin scaffold offers many advantages when compared to immunoglobulin based tools, because avidin is thermally (T _m = 83.5 ⁰ C, T _m = 117 ⁰ C (with biotin)) and chemically stable protein (Ross et al., 1986; Nordlund et al., 2003) and avidin variants are efficiently produced in bacterial cells. Avidin forms with increased stability have also been developed (Nordlund et al., 2003; Hytδnen et al. 2005b).

Since the ligand-binding site of avidin is a deep cavity, it offers an interesting alternative to antibodies as a template to bind small ligands. Avidin naturally binds tightly water soluble small vitamin H, biotin, which has molecular weight of 244.31 g/mol. Therefore, avidin is structurally well-adapted base for development of novel binders for other small ligands.

To broaden the possible use of avidin, it would be beneficial to have avidin forms specifically binding other ligands than biotin. To achieve this, one would need to have a high through-put method to screen active binders from a collection of mutagenized avidin forms. Phage display methodology is shown to be extremely powerful technique for such purposes. Here we have developed phage display method for avidin and used it to capture avidin mutants with affinity towards testosterone.

SUMMARY OF THE INVENTION

The present invention is directed to a method for producing from a chicken avidin (SEQ ID NO: 1) a modified avidin polypeptide binding to a small molecule other than biotin, the method comprising the steps of:

a) preparing a library of bacteriophages displaying multiple modified avidin polypeptides with randomized mutations in the loop 1-2 and/or loop3-4 sequence, i.e. amino acid residues 12-16 and/or 35-38 of SEQ ID NO:1, respectively;

b) selecting from said library those bacteriophages displaying modified avidin polypeptides which bind to a small molecule of interest;

c) isolating and expressing a small-molecule-binding modified avidin polypeptide obtained from step b).

The present invention is also directed to a modified avidin polypeptide comprising essentially the sequence of SEQ ID NO: 1 in which the loop of amino acid residues 12-16 of SEQ ID NO: 1 connecting beta strands 1 and 2 is modified and said avidin polypeptide specifically binds to a small molecule other than biotin. Further, the present invention also describes modified avidin polypeptides comprising any of the sequences selected from the group consisting of SEQ ID NOS :21-29 in which the loop of amino acid residues 35-38 of SEQ ID NO:1 connecting beta strands 3 and 4 are modified and said avidin polypeptides specifically bind to a small molecule other than biotin.

Further, the present invention provides modified avidin polypeptides binding to testosterone and nucleic acid vectors and host cells expressing or secreting said modified avidin polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic presentation of the avidin phagemid expression vector constructs. The phagemid constructs for Avd (A), double Avd (B) and dcAvd (C) phage display. Phagemid construct for avidin (A) is used in loop library application (Example C). The phagemid vector contains the lac promoter and the f^origin. The pelB signal sequence is used for secretion of the avidin-pIII fusions and the free avidin of the double avidin display construction (B). The restriction enzyme cleavage sites used are shown by vertical arrows.

Figure 2. Immunoblot analyses of avidin phages with avidin (A) and pill (B) antibodies. The samples in the immunoblots are 1) Avd phage, 2) Avd(N118M) phage, 3) Avd double construct phage and 4) Avd(N118M) double construct phage. Avidin-pIII fusion proteins are indicated by black arrows. Theoretical mass of the avidin-pIII fusion protein is 38 kDa which is in good agreement with the apparent size of the band in the immunoblot. In the double display constructs (samples 3 and 4 in panel A), free avidin (-14 kDa) is produced in addition to the avidin-pIII fusion. This is indicated with dark grey arrow (lower arrow in panel A). The unfused full-length pill migrating at -60 kDa is indicated with dark grey arrow (sample 1 in panel B) and the C-terminal domain of the pill (-35 kDa) is indicated with light grey arrow (the lowest arrow in panel B). Figure 3. Randomization of avidin sequence

A) A simplified structural representation of the loop randomization strategy for an avidin monomer. Four to six mutagenized residues are shown as yellow spheres in each modified loop. B) Topological diagram of the strategy.

Figure 4. A schematic illustration of the production of DNA library encoding mutagenized avidins.

For each of the three loops of avidin in which the amino acid are to be mutated, an oligodeoxynucleotide was synthesized (Fig 5). For 1-2 loop library a nucleic acid fragment of 105 base pairs was amplified (Step 1, A) with the primers Avd_NheI_5' and Loop 1-2 _R1_3' (schematically referred in the figure as Avd_mutant_3') using wt avidin cDNA as a template. Parallel to this, a nucleic acid fragment with 357 base pairs was PCR- amplified (Step 1, B) with the primers Loop 1-2_R2_5' (schematically referred in the figure as Avd_combine_5') and Avd_NotI_3', also using wt avidin as a template. The mixture of both of these fragments served as a template in a second amplification step in the presence of PCR primers Avd_NheI_5' and Avd_NotI_3', wherein a DNA fragment of 462 base pairs was obtained. For 3-4 loop library a nucleic acid fragment with 166 base pairs was amplified (Step 1, A) with the primers Avd_NheI_5' and Loop 3-4 _R1_3' (schematically referred in the figure as Avd_mutant_3') using wt avidin cDNA as a template. Parallel to this, a nucleic acid fragment with 285 base pairs was amplified (Step 1, B) with the primers Loop 3-4_R2_5' (schematically referred in the figure as Avd_combine_5') and Avd_NotI_3', also using wt avidin as a template. The mixture of both of these fragments served as template in a second amplification step in the presence of PCR primers Avd_NheI_5' and Avd_NotI_3', wherein a DNA fragment of 451 base pairs was obtained. For 5-6 loop library a nucleic acid fragment with 271 base pairs was amplified (Step 1, A) with the primers Avd_NheI_5' and Loop 5-6 _R1_3' (schematically referred in the figure as Avd_mutant_3') using wt avidin cDNA as a template. Parallel to this, a nucleic acid fragment with 178 base pairs was amplified (Step 1, B) with the primers Loop 5-6_R2_5' (Avd_combine_5') and Avd_NotI_3', also using wt avidin as a template. The mixture of both of these fragments served as template in a second amplification step in the presence of PCR primers Avd_NheI_5' and Avd_NotI_3', wherein a DNA fragment of 449 base pairs was obtained. Figure 5. Oligonucleotides used in the avidin phage display and in the loop library DNA randomization.

Oligonucleotides with randomized region (NNN) correspond to Avd_mutant_3' oligonucleotide in Figure 4. Oligonucleotides Loop 1-2_R2_5', Loop 3-4_R2_5' and Loop 5-6_R2_5' correspond to Avd_combine_5' oligo in Figure 4. Sequences of the oligonucleotides are chosen so that stop codons can not arise in the region of the mutated sequence.

Figure 6. Avidin variants selected from the phage library expressing avidins with randomized loop between beta strands 1 and 2 using testosterone as a capture ligand. Amino acid residues 4-22 of avidin are presented here. The sequences of four avidin mutants showing testosterone binding are shown (mutant 1-4, i.e. only altered part of SEQ ID NOS:2-5 are shown). The secondary structure elements in wild-type avidin (SEQ ID NO. 1; coordinates for 3D-structure from Protein Data Bank accession code 2AVI) are shown above the alignment.

Figure 7. Microplate analysis of avidin-displaying phages.

Attachment of avidin-displaying phages on the surface of microplate coated with particular ligand were studied. The background signal coming from non-spesific binding is subtracted from the results shown here. A) As expected, wt Avd phages, both single and double constructs, gave stronger absorbencies (indicating tighter binding) when biotin coated surface was used. When using HABA coated surface Avd(N118M) phages, both single and particularly double constructs, gave stronger absorbencies compared to wt Avd phages. This might be due to the more tight binding of the mutant than the wt Avd to the HABA surface. 1:2 and 1:5 refers to the dilution of the phages. B) dcAvd phages were introduced to biotin surface to detect functionality of dimeric avidin on the displayed on the phage surface.

Figure 8. Binding of avidin variant (SEQ ID NO. 2) to testosterone-coated surface in optical biosensor analysis.

Binding of avidin variant (SEQ ID NO. 2, c = 1 μM) to Biacore X sensor chip functionalized with testosterone-BSA. The binding was measured in the presence of varying concentrations (0-50 μM) of inhibiting testosterone. After injection of the sample, the sensor surface was washed by running buffer.

Figure 9. Inhibition of binding by various steroid molecules.

Binding of avidin variant (SEQ ID NO. 2, c = 1 μM) to Biacore X sensor chip functionalized with testosterone-BSA. The binding was measured in the presence of varying inhibitors (50 μM). After injection of the sample, the sensor surface was washed by running buffer.

Figure 10. Microplate assay for determination of ligand binding activity of avidin mutants selected by phage display

Nine avidin forms (SEQ ID NOS :21-29) selected by phage display were analyzed by microplate assay in which the binding of avidin 3 to 4 loop mutant proteins extracted from E. coli cells to microplate-immobilized ligands were measured. (N.D. = Not determined), see also Table 1. Binding of avidin variants was measured with BSA conjugated with three different steroid hormones (testosterone, DHEAS and DHT). Additionally, the binding was competed in the presence of the free ligand (50 μM testosterone, DHEAS or DHT).

DETAILED DESCRIPTION OF THE INVENTION

Avidin naturally binds small water-soluble vitamin H, biotin. The binding site is at the one end of the eight-strand beta-barrel. The tight and specific biotin-binding capacity of avidin is employed in wide array of biotechnology applications.

In order to develop novel binding characteristics, a phage display methodology has been developed for chicken avidin. The system involves bacterial signal peptide, which makes it possible to produce active avidin molecules in E. coli cells. Avidin is fused to phage protein pill in the developed procedure. Randomized mutagenesis of avidin is subjected to loop regions surrounding the ligand-binding site. Selection of active binders is performed by immobilizing the ligand of interest on a surface and by selecting bacteriophages expressing avidin variants binding the particular ligand by use of the activated surface.

The present invention relates to method which allows functional display of avidin on the surface of M 13 phage fused to the C-terminal region of the pill protein. Earlier it has been shown that avidin is efficiently expressed in soluble form in E. coli when periplasmic secretion was achieved by N-terminal bacterial signal peptide (Hytδnen et al., 2004). Production of active protein in bacteria is essential for functional display on phages. Phage display methodology has been used for efficient selection of modified proteins (Skerra, 2007).

Three different strategies to display the avidin scaffold in an active form on the M 13 phage were evaluated (Example A). In the first display construct the Avd is produced solely as a pill fusion (Fig. 1 A). In the double Avd display construct, in addition to the pill-fused avidins, also the free Avd monomers were produced (Fig 1 B). This should lead to more efficient assembly of the tetrameric avidin scaffolds, especially if membrane anchoring of avidin by pill fusion partner causes negative effect for oligomerization. We suppose that the oligomerization of avidin secreted by the pelB signal sequence takes place in the periplasmic space of E. coli allowing the display of functional avidin on the phage, as in the case of antibody Fab scaffold display requiring folding of two chains for functional antibody molecule (Hoogenboom et al., 1991). The third avidin display construct was based on the dcAvd scaffold (Fig. 1 C). All avidin phages yielded routinely titers of 10 ¹² cfu/ml.

The phages displaying different avidins were analysed by immunoblotting from phage stocks (Fig 2). The theoretical mass of avidin-pIII fusion protein is 38 kDa, which agrees well with protein size detected with anti-avidin in the immunoblot analysis (-38 kDa) (Fig 2 A). The immunoblot analysis indicates that avidin-pIII fusion, as well as the free avidin expressed from the double avidin display construct, can be readily identified, but the dcAvd-pIII fusion is expressed with lower efficiency. The control avidin from chicken eggs (Belovo, Liege, Belgium) is glycosylated, and therefore it migrates to the position somewhat upper than bacterially expressed, nonglycosylated avidin from phages. The immunoblot analysis also showed that the avidin-pIII fusion is partially proteolytic ally cleaved, since a band migrating at the position of C-terminal pill fusion (-35 kDa) was detected with the anti-pill antibody (Fig. 2B). In phage display the whole pill (migrating at -58 kDa) is also produced besides the fusion and was detected in the immunoblot with the anti-pill antibody. The theoretical mass of dcAvd-pIII fusion protein is 54 kDa, which species were observed in the immunoblot analysis (not shown).

Ligand-binding properties of avidin phages were analyzed with microplate-based experiments. As expected the wt avidin phages gave stronger absorbencies than the phages displaying Avd(N118M), when bound to biotin surface, which indicates higher binding affinity of wt avidin as compared to the mutant (Fig. 7 A). In contrast, when attached to HABA-coated surface the Avd(Nl 18M) phages gave much stronger absorbencies than wt avidin (Fig. 7 A). This indicates most probably more tight binding of the Avd(Nl 18M) phages to the HABA surface as compared to phages displaying wt avidin (EXAMPLE B). Furthermore, phages carrying double constructs gave higher signal than phages carrying single constructs. This might be due to the more tight binding of displayed avidins (affinity) or alternatively increased number of binding sites per phage (avidity). Also it is possible that there are more functional avidins on the phage surface due to increased folding efficiency in presence of free subunits. However, difference between single and double construct phages was not as large as would be expected. The reason for that could be oligomerization of single avidin constructs on the phage surface. It is known that oligomerization of avidin increases the binding affinity of avidin ligands due to architecture of the binding site (Livnah et al., 1993).

In order to develop a library of avidin mutants with capability to bind ligands other than avidin, loop regions close to the ligand-binding site were subjected to random mutagenesis. This strategy minimizes the perturbation of the structural integrity of avidin since the beta-strands forming the eight-stranded beta-barrel are not modified. Furthermore, only one end of the barrel is modified. Loop regions between beta strands 1 & 2, 3 & 4 and 5 & 6 were subjected to randomization in separate libraries. This can be achieved by DOGS methodology, for instance (Gibbs et al., 2001). Therefore, the present invention is directed to a method for producing from a chicken avidin (SEQ ID NO: 1) a modified avidin polypeptide binding to a small molecule other than biotin, wherein any of the above-mentioned loop regions is modified. As an example of functionality of avidin loop libraries, an avidin library with randomized 1-2 loop region (five randomized amino acid residues) was biopanned against BSA- conjugated steroids. Interestingly, avidin mutants were detected to be enriched after several selection rounds with testosterone-coated surfaces (Figure 6). From these enriched mutants (SEQ ID NO. 2) was selected for further analysis and affinity maturation (EXAMPLE C, EXAMPLE D)

In affinity maturation avidin polypeptides specifically binding immobilized testosterone were screened from gene library, where the DNA sequence encoding amino acid residues 35-38 of SEQ ID NO:2 in the loop connecting beta strands 3 and 4 was randomized. From the mutants detected in the screening, nine mutants (SEQ ID NO:21-29) were selected for further analysis. Clear improvement on testosterone binding (affinity maturation) was observed based on microplate analysis (Figure 10, Table 1).

The DNA encoding avidin mutant (SEQ ID NO. 2) was subcloned from phagemid vector to petlOlD/TOPO expression vector by using polymerase chain reaction. DNA sequence encoding signal peptide ompA, enabling production of active avidin in E. coli (Hytδnen et al., 2004), was attached to the 5' end of the avidin cDNA. Furthermore, DNA encoding polyhistidine tag was attached to the 3 'end of the sequence (EXAMPLE E).

The expression of the avidin mutant in E. coli BL-21(AI) was done essentially as described in (Hytδnen et al., 2004) at 26 ⁰ C temperature (EXAMPLE E). The protein was isolated using Ni-NTA sepharose (Qiagen) according to instructions from the manufacturer (EXAMPLE E). Analysis with SDS-PAGE revealed a band with correct molecular weight. Gel filtration analysis showed that the expressed protein forms tetramers, like those of wt avidin.

Use of engineered avidins with novel binding characteristics in combination with dual chain and single chain avidin technology may allow one to develop avidins capable to bind multiple ligands simultaneously (Nordlund et al., 2004; Hytδnen et al., 2005a; Nordlund et al., 2005). These kinds of proteins would be valuable molecules to clinical and diagnostic applications, as avidin protein has some benefits, such as high thermal and chemical stability, when compared to antibodies. Furthermore, production of antibodies towards small molecules has proven to be difficult, whereas the binding site of avidin is designed for a small ligand. Avidin scaffolds with affinity towards novel ligands could replace antibodies for example in diagnostic applications that face more demanding conditions.

The term "specifically binding" refers herein to a low background and high affinity binding between the modified avidin polypeptide of the invention and its target molecule (i.e. lack of non-specific binding).

The term "small molecule" refers herein to small analytes such as steroids, hormones, metabolites, therapeutic and abused drugs, environmental pollutants and toxins. The molecular weights of these small analytes are normally less than 5000, but the limits are not absolute.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The invention will be further described with reference to the following non-limiting examples.

EXAMPLES

EXAMPLE A: Avidin display on M13 bacteriophage

Appropriate restriction sites were added to cDNA of avidin core (Gope et al., 1987) (lacking the DNA sequence encoding the signal peptide of 24 N-terminal amino acids) by PCR. 100 ng of pVTTphagemid plasmid DNA were employed as template and 100 pmol of each primer were added. In addition, the reaction mixture contained 5 μl lOxDynazyme buffer F-511 with MgSO ₄ , 4 μl dNTP-mix (2 mM dATP, dCTP, dGTP, dTTP). Purified water was added to the mixture so that the final volume of 50 μl was achieved. Subsequently 1.5 units of Dynazyme DNA-polymerase (Finnzymes) was added. PCR was performed as follows: reaction was first heated to 95 ⁰ C for 2 minutes, which was followed by 24 temperature cycles of 30 sec at 94 ⁰ C, 30 sec at 55 ⁰ C and 1 minute at 72 ⁰ C. After temperature cycling reaction was incubated for 10 minutes at 72 ⁰ C. The desired amplification products (-450 bp) were isolated from analytical grade agarose (Promega) by agarose gel electrophoresis using the Nucleo Spin Extract II kit (Macherey-Nagel) according to the instructions of the manufacturer. PCR product was subcloned into the pCR ^® 2.1-TOPO plasmid by TOPO TA-cloning (Invitrogen) and and the cloning reaction mixture was transformed into TOPlO cells according to the instructions of the manufacturer. Plasmids were extracted from the colonies selected based on the blue- white screening. The nucleotide sequences of the avidin pCR ^® 2.1- TOPO plasmid constructions were verified by an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the protocols recommended by manufacturer (ABI PRISM BigDye Terminator Cycle Sequencing Kit v.1.1, Applied Biosystems). Avidin fragments were cut out from the TOPO-plamid with appropriate restriction enzymes and cloned into the phage display vector (pVTTphagemid). The phage display vector contains the lac promoter (plac), the signal sequence of pectate lyase protein (pelB) from Erwinia carotovara (Lei et al., 1987), transcription terminator region and the phagemid origin of replication (f ⁴ ^). The cDNA of avidin core (Gope et al., 1987) was cloned into phagemid vector as a N-terminal fusion to the C-terminal domain (amino acids 198-406) of the minor phage coat protein III. Restriction sites to avidin was added by using primers

Avd_NheI_5' and Avd_NotI_3' (Fig. 5) in PCR reaction mentioned above. C-terminal part of the pill has theoretical molecular weight of 23 kDa (amino acid residues 198- 406). The construct expressing Avd(Nl 18M) fused to phage protein III fragment was prepared identically.

The other avidin display phagemid construction was designed to express both free avidin and avidin-pIII fusion. This should facilitate the assembly of functional, tetrameric avidin on the phage surface, since pill fusion partner presumably negatively affects the oligomerization of avidin. In these constructs, the coding sequence of the free avidin was subcloned using primers Avd_NheI_5' and Avd_AscI_stop_3' (Fig. 5). The second expression construct was prepared with primers Avd_SfiI_5' and Avd_NotI_3' and subsequently cloned to pVTTphagemid. Analogous strategy was used to insert the DNA encoding Avd(N118M) blocks to the vector.

The third avidin display system studied employed the previously described dual chain avidin (Nordlund et al., 2004). In this strategy dual chain avidin was cloned directly into the phagemid vector. First, one circularly permuted avidin (cpAvd5→4; described in (Nordlund et al., 2004)) was cloned into the phagemid vector using primers cp54Avd_NheI_5' and cp54Avd_BamHI_NotI_3'(Fig. 5). In the second step, another circularly permuted avidin (cpAvd6→5; described in (Nordlund et al., 2004)) was cloned to this construct using primers Single2 and cp65Avd_NotI_3'(Fig. 5), employing the introduced BamHI site in cpAvd5→4.

The nucleotide sequences of the avidin phagemid constructions were verified by an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) according to the protocols recommended by manufacturer (ABI PRISM BigDye Terminator Cycle Sequencing Kit v.1.1, Applied Biosystems). The avidin display constructions used in this study are presented in Figure 1.

Phagemid vectors including avidin were transformed into chemically competent E. coli XL 1 Blue cells (Stratagene, La Jolla, CA) with the heat shock method. Phage stocks of the different avidin and Avd(Nl 18M) display constructs were made from individual colonies picked from the transformation plates into Super broth (SB) medium supplemented with 100 μg/ml ampicillin, 10 μg/ml tetracycline and 2% glucose. The o/n seed cultures grown at 37°C were diluted 1:50 into 10 ml of SB supplemented with 100 μg/ml ampicillin and 10 μg/ml tetracycline. The cultures were grown to ODβoo ~1, where after they were infected with the helper phage (10 ¹² pfu) VCSM-M 13 (Stratagene). The infected cultures were incubated for 30 min at 37°C and then diluted to the final volume of 100 ml of SB medium supplemented with 100 μg/ml ampicillin and 10 μg/ml tetracycline. The cultures were grown for 2 h at 37°C in a shaker (250 rpm). Kanamycin was added to the cultures to the final concentration of 70 μg/ml, where after the cultures were grown for ~ 16 h at 34 °C in a shaker (250 rpm). The cultures were centrifuged (4000 g, 15 min, at 4°C) and the phages were precipitated from the supernatant by adding 25 ml of 20% PEG, 2.5 M NaCl (PEG/NaCl) and incubation for 30 min on ice. PEG precipitated phages were centrifuged at 4 °C with 13000 g for 20 min. The phage pellets were resuspended into 2 ml of PBS. Bacterial cell debris was removed by centrifugation at 4°C with 13 000 g for 5 min, and phages were re-precipitated by adding 200 μl of PEG/NaCl to the supernatant. After 15 min incubation on ice, phages were pelleted by centrifugation at 4°C with 13 000 g for 5 min. Phage pellets from were resuspended into PBS or PBS + 0.5% BSA and stored at 4 ⁰ C. Purified phages were analysed with SDS- PAGE and western blotting following immuno staining with anti-avidin (University of OuIu, 1:5000) and with anti-pill antibody (Biosite, Sweden, 1:2000) (Fig. 2). EXAMPLE B: Functionality of displayed avidin forms assayed by microplate method

The affinity of phage avidin towards biotin and HABA were analyzed with microplate experiment. Maxisorp immunoplates were coated with biotinylated insulin (10 μg/ml) and with HABA-BSA conjugate (10 μg/ml). Phages suspended in PBS with 0.5% BSA (PBS-BSA; 10 ¹² cfu/ml, 100 μl) were diluted with PBS-BSA (Dilutions 1:2 and 1:5) and added to the coated wells and incubated for 20 minutes, where after the wells were washed five times with PBS-Tween (0.05% v/v Tween 20) and ten times with PBS. To prevent non-spesific binding 100 μl of PBS-BSA were added to the wells. After 10 minutes incubation wells were washed three times with PBS and then the HRP/M13 phage coat antibody (Amersham biosciences, RPAS Detection module) (dilution 1:5000, 100 μl) was added. After 10 min incubation at room temperature the wells were washed three times with PBS-Tween and five times with PBS. The ABST (2,2'-azino-bis-(3- benzthiazoline-6-sulfonic acid)) substrate solution with hydrogen peroxide was added into the wells and absorbance was read at 405 nm after 30 min. The binding of the helper phage (VCSM 13) to biotinylated insulin was used to analyze the level of the unspecific binding. The results are summarized in figure 7.

EXAMPLE C: Construction of a loop library for avidin

PCR was applied in multiple steps according to FIG. 4 for concerted mutagenesis of in total 15 amino acids in different positions in the three loops of avidin. PCR reactions were carried out in a volume of 100 μl in all amplification steps. In both of the first amplification steps 100 ng of pVTTphagemid plasmid DNA were employed as template as well as 100 pmol of the respective primers. In addition, the reaction mixture contained 10 μl lOxPfu buffer with MgSO ₄ , 2 μl dNTP-mix (2 mM dATP, dCTP, dGTP, dTTP). Purified water was added to the mixture so that the final volume was achieved. Subsequently 2.5 units of Pfu DNA-polymerase (Fermentas) was added. For the amplification step where PCR product is < 200bp, PCR-program was performed as follows: first reaction was heated to 95 ⁰ C for 1 minute, this was followed by 35 temperature cycles of 40 sec at 95 ⁰ C, 30 sec at 54 ⁰ C and 2.5 minutes at 72 ⁰ C. Reaction was then incubated at 72 ⁰ C for 5 minutes. For the amplification step where PCR product was > 200bp, PCR-program was performed as follows: first reaction was heated to 95 ⁰ C for 1 minute, this was followed by 35 temperature cycles of 45 sec at 95 ⁰ C, 40 sec at 53 ⁰ C and 2 minutes at 72 ⁰ C. Finally, the reaction was incubated at 72 ⁰ C for 5 minutes. The reaction was analyzed by agarose gel electrophoresis (analytical grade agarose, Promega) and the desired reaction product was isolated using the Nucleo Spin Extract II kit (Macherey-Nagel) according to the instructions of the manufacturer.

In the subsequent amplification step 100 ng of both of PCR fragments (A+B) were used as a templates, and 50 pmol of each primers Avd_NheI_5' and Avd_NotI_3'were added to reaction. The remaining components of the PCR mixture were used in the same amount as in the previous amplification steps. PCR took place with first heating up to 95 ⁰ C for 1 minute and this was followed by 35 temperature cycles of 50 sec at 95 ⁰ C, 50 sec at 52 ⁰ C and 170 sec at 72 ⁰ C. After temperature cycling, the reaction was incubated for 5 minutes at 72 ⁰ C. The expected fragment was again isolated by agarose gel electrophoresis.

This fragment (-450 bp) was cut with restriction enzymes Nhel (Fermentas) and Notl (Fermentas). The purification of nucleic acid after digestion was performed using the Nucleo Spin Extract II kit (Macherey-Nagel) according to the instructions of the manufacturer. The DNA of the vector pVTTphagemid was analogously cut with Nhel and Notl and the larger of the two fragments was isolated (3500 bp).

For the ligation, 1 μg of the PCR fragment and 1 μg of the vector fragment were incubated in the presence of 50 Weiss Units T4 DNA ligase (Fermentas) in a total volume of 200 μl for 14 hours at 16 ⁰ C. The DNA was precipitated by adding 20 μl 3 M sodium acetate and 200 μl cold ethanol. Incubation at -20 C for 30 minutes was followed by centrifugation (30 minutes, 15000 g, 4 ⁰ C). The pellet was washed with 1000 μl ethanol (70% v/v, -20 ⁰ C) and dried in the hood. The pellet was then dissolved in 10 ul Of MgCl ₂ .

The electroporation was carried out to transform electrocompetent E. coli XL 1 Blue (Stratagene) cells according to the instructions of the manufacturer. BioRad cuvettes

(electrode separation 2 mm) was used (2.5 kV/cm, 200 Ω, 25 μF) for electroporation. 4 μl of the ligation mixture was added to 40 μl of the cell suspension and this was transferred to the cuvette. After the electroporation the suspension was immediately diluted in 960 μl of warm SOC medium (2% w/v tryptone, 0.5% yeast extract, 10 mM NaCl, 10 mM MgSO ₄ , 10 niM MgCl ₂ ) and was shaken for 60 minutes at 37 ⁰ C and 220 rpm. In total 5 μg of the ligated DNA/loop library was transformated and Ix 10 ⁵ transformants were obtained after plating to LB agarose supplemented with ampicillin (50 μg/ml). The colonies were collected from the plates with glycerol and stored as glycerol stock at - 7O ⁰ C .

EXAMPLE D: Selection of avidin mutants binding testosterone by avidin-displaying bacteriophages

Glycerol stock of E. coli cells was added to the growing medium and the culture was incubated at 37 ⁰ C with 250 rpm shaking to obtain cell density of OD600 = 0.8-1. The culture was then infected with VCS-M 13 helper phage (Stratagene) and shaken for additional 1 h at 37 ⁰ C, 250 rpm. Kanamycin (70 μg/ml) was added and the incubator temperature was lowered to 34 ⁰ C. Incubation continued over night at 34 ⁰ C with shaking at 250 rpm.

In the following day, the infected cells were sedimented by centrifugation (15 minutes, 5000 rpm, 4 ⁰ C). The supernatant containing the phage particles was mixed with ¹ A volumes 20% w/v PEG 8000, 15% NaCl and was incubated for 30 minutes at 4 ⁰ C. After centrifugation (20 minutes, 9000 rpm, 4 ⁰ C) supernatant was discarded and pellet was suspended in 2 ml of PBS and centrifuged for 10 minutes at 4 ⁰ C, 13000 rpm. ¹ A volumes w/v PEG 8000, 15% NaCl was added to supernatant and mixed carefully. After incubation for 15 minutes on ice, mixture was centrifuged for 10 minutes at 4 ⁰ C, 13000 rpm. Supernatant was discarded and pellet was suspended to 1 ml of PBS (4 mM KH ₂ PO ₄ , 16 mM Na ₂ HPO ₄ , 115 mM NaCl, ph 7.4). Phages were store at 4 ⁰ C.

For selection of active binders, NUNC immunosorp plates were coated overnight with testosterone-BSA conjugate (TES-3-CMO-BSA, Sigma, 5 μg/ml) and with insulin biotin (Sigma, 5 μg/ml) and blocked by incubating for 1 h at room temperature with 0.5% PBS- BSA. The wells were then washed three times and fresh phages were first added to the insulin biotin-coated wells (100 μl of phages/well) and incubated for 15 minutes at room temperature on a shaker. Subsequently phages were transformed to testosterone-coated wells and incubated for 15 minutes at room temperature on a shaker. For the removal of non-bound phages, the wells were washed using the following protocol: During the first selection round the washing was performed three times with PBS-Tween 20 (0.05% v/v) and five times with PBS. Bound phages were eluted by 10 minute treatment with a solution of 100 mM HCl containing biotin (50 μg/ml) with vigorous shaking. After 10 minutes incubation, the mixture was neutralized by adding 5 μl of 2 M Tris solution. For the next selection rounds, the eluted phage pools were amplified by infecting exponentially growing E.coli XLl-Blue cells. Three to four further selection cycles with TES-3-CMO-BSA conjugate were carried out in this way. Washing conditions were stringed up in every panning rounds to diminish unspecific binding. During the second panning round the washing was performed three times with PBS-Tween 20 (0.05% v/v) and five times with PBS. During the third and following panning rounds the washing was performed five times with PBS-Tween 20 (0.05% v/v) and ten times with PBS.

The quality of the phage library was evaluated between selection rounds by using restriction enzymes followed by analysis in agarose gel electrophoresis. Several clones from each selection round were subjected to DNA sequencing.

EXAMPLE E: Production of avidin mutant selected by phage display

The gene cassette between Nhel and Notl cleavage sites from the VTTphagemid vector was extracted and used as a template in PCR where region encoding histidine tag (6xHis) was added to the 3 'end of the protein-encoding region, followed by stop-codon. Furthermore, a CACC sequence was added to the 5' end of the PCR- to aid directional TOPO ^® cloning (Invitrogen). PCR-program was performed as follows: first reaction was heated to 95 ⁰ C for 2 minutes, this was followed by 35 temperature cycles of 1 minute at 95 ⁰ C, 1 minute at 52 ⁰ C and 3 minutes at 72 ⁰ C. After temperature cycling the reaction was incubated for 5 minutes at 72 ⁰ C. The desired amplification product (-450 bp) was isolated from analytical grade agarose (Promega) after agarose gel electrophoresis using the Nucleo Spin Extract II kit (Macherey-Nagel) according to the instructions of the manufacturer and subsequently ligated the expression plasmid pET101/D. Ligation was transformed to the E. coli TOPlO cell line by heat shock method followed by plating to LB-agar plates containing 50ug/ml ampicillin. Individual clones were picked from the plates and subjected to small-scale liquid culturing. Plasmids were isolated form o/n cultures and subjected to DNA sequencing. Verified constructs were then used for transformation of E. coli BL21-AI cells (Invitrogen) and plated to LB-agar plates containing 50 ug/ml ampicillin. After o/n incubation at 37 ⁰ C, individual colony was transferred to 5 ml LB supplemented with tetracyclin (10 μg/ml) and ampicillin (100 μg/ml) and cultured o/n at 26 ⁰ C. Small culture was then used as a seed for 500 ml culture in which the protein expression was induced by arabinose when OD (measured at 600 nm) reached 0.2-0.4. Purification of the avidin mutant from E. coli cells was lysed by sonication (Sonics & Materials Vibra Cell™, VC 505, 500W, 5 sec on, 2 sec off, program was performed two times with amplitude 50%) and DNaseI (New England Bio Labs) was added after sonication (3 μl of DNase (20 mg/ml) with 20 μl of 1 M MgSO4, incubate 1 h at 37 ⁰ C). Purification of proteins was performed using Ni-NTA affinity chromatography according to the instructions of the manufacturer (Qiagen).

EXAMPLE F: Analysis of avidin mutant by SPR method

Binding of the purified wild-type avidin and avidin mutant SEQ ID NO: 2 to testosterone-BSA conjugate (TES-3-CMO-BSA, Sigma) immobilized on an dextran- coated sensor chip (Biacore CM5 chip) were determined by Biocore X instrument (BIAcore, GE Healthcare, Uppsala, Sweden). The dependence of the surface plasmon resonance response on the concentration of the purified avidin proteins injected on the biosensor surface indicated moderately tight binding between avidin mutant SEQ ID NO. 2 and immobilized ligand. The ligand, TES-3-CMO-BSA, was coupled to the CM5 sensor chip through amine coupling using EDC as crosslinker. The measurement was carried out in 50 mM Na-phosphate pH 7.0 containing 1 M NaCl. In these assay conditions, wild-type avidin (SEQ ID NO: 1) showed no binding to testosterone- functionalized surface, whereas avidin mutant (SEQ ID NO: 2) showed clear binding in concentration of 1 μM and above (calculated for monomer). The presence of testosterone inhibited the binding (FIG. 8). Also, addition of biotin caused inhibition of the binding (not shown), which indicates that the binding site of testosterone is located close to the bio tin-binding site.

In order to study the specificity and binding activity of the avidin mutant set forth in SEQ ID NO: 2, a binding reaction was measured in the presence of various steroid molecules (FIG. 9). Among the group of steroids analyzed, estradiol (EST) and dihydrotestosterone showed the weakest inhibition of binding, indicating lowest affinity towards avidin mutant SEQ ID NO: 2. Testosterone (TES), dehydroepiandrosterone (DHEAS) and androstenedione (ANS) showed strong inhibition, suggesting their high affinity towards the avidin mutant SEQ ID NO: 2. Biotin (BTN) inhibited the binding efficiently.

EXAMPLE G: Affinity maturation of testosterone binding

Library was constructed as was shown in EXAMPLE C with the following exceptions: the sequence of SEQ ID NO:2 was used as a template in PCR. DNA sequence encoding amino acid residues 35-38 were randomized in the PCR reaction (primer new_3_4_R_l_3' is used instead of 3_4_R_1_3')- These residues are located in the loop connecting beta strands 3 and 4 in avidin. A phage library was generated as described in EXAMPLE C. The constructed phage library was screened as described in EXAMPLE D with the following exceptions: All overnight incubations were performed at 28 ⁰ C. During biopanning step before adding phage pool into coated wells, a fraction of phage pool was preincubated with 36 μM DHT (dihydro testosterone) for 10 min. Bound phages were eluted from immobilized testosterone by 10 minute treatment with a solution of 100 mM HCl containing either testosterone (50 μg/ml) or 1:1 mixture of DHEAS (dehydroepiandrosterone) and androstenedione. This treatment was done with vigorous shaking. After 10 minutes incubation, the mixture was neutralized by adding 5 μl of 2 M Tris solution. Several clones from every panning round were screened with microplate assay either by measuring the binding of phage displaying avidin-pIII protein or by measuring the binding of avidin-pIII protein extracted from E. coli periplasm to immobilized testosterone-BSA, following determination of bound avidin-pIII by using polyclonal avidin antibody as described in EXAMPLE H. The following avidin mutants were selected based on the results of the screening: SEQ ID NO:21-29.

EXAMPLE H: Determination of ligand-binding properties of affinity-maturated avidin mutants by microplate assay

The selected avidin mutants (SEQ ID NO:21-29) were produced for further analysis as described in EXAMPLE E. Maxisorp immunoplates were coated with steroid hormone- BSA conjugate (500 ng) (DHEAS, DHT, testosterone). After washing wells were blocked with 100 μl of 5% milk in 50 niM Tris-HCL buffer 1 M NaCl pH7.9 (30 min incubation). A control sample of protein was preincubated with corresponding free ligand (50 μM) for 2 h. After blocking step, 2.5 μg of mutant avidin protein diluted in 50 mM Tris-HCL buffer 1 M NaCl pH7.9 was added into the wells and incubated for 2 h. Wells were washed three times with PBS-Tween (0.05% v/v Tween 20) and five times with PBS. A solution containing primary antibody (anti-avidin, University of OuIu) was then added (dilution 1:5000, 100 μl) and incubated for 1 h. Wells were washed three times with PBS-Tween (0.05% v/v Tween 20) and five times with PBS. A solution of secondary antibody (GAR-AP) was then added (dilution 1:5000, 100 μl). After 1 h incubation wells were washed three times with PBS-Tween and five times with PBS. The NBT/BCIP substrate solution within DEA buffer was added into the wells and absorbance was read at 405 nm after 60 min incubation. The results are summarized in Figure 10 and Table 1.

Table 1.

Protein

Inhibition Inhibition Inhibition with free with free with free

Amino acids Testosterone testosterone DHEAS DHEAS DHT DHT

35-38 A405 A405 A405 A405 A405 A405

SEQ ID NO: 2 0.149 0.061 0.063 0.042 0.046 0.048

LLRG 1 .199 0.684 0.926 0.673 0.812 0.640

ATVN 1 .737 0.181 0.384 0.145 0.400 0.083

VYDY 0.009 0.016 0.000 0.014 0.000 0.007

VVTH 0.055 0.025 N. D. N. D. N. D. N. D.

ITAS N. D. N. D. N. D. N. D. N. D. N. D.

YILP N. D. N. D. N. D. N. D. N. D. N. D.

LYSL N. D. N. D. N. D. N. D. N. D. N. D.

SFPS N. D. N. D. N. D. N. D. N. D. N. D.

VNPV N. D. N. D. N. D. N. D. N. D. N. D.

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