Abstract:
The present invention relates to new chimeric mutants of avidin protein with improved properties, e.g. thermostability, better stability against proteolysis, better charge properties (for example lower pl) compared to native avidin and avidin-related proteins, AVRs. The chimeric avidin mutants comprise mutants where a region or regions in avidin are substituted by a corresponding region or regions from an AVR protein.

Description:
FIELD OF THE INVENTION 
     The present invention relates to new chimeric mutants of chicken avidin protein with improved properties, e.g. thermostability, better stability against proteolysis, better charge properties (for example lower pl) compared to native avidin and avidin-related proteins, AVRs. The chimeric avidin mutants comprise mutants where a region or regions in avidin are substituted by a corresponding region or regions from an AVR protein. 
     BACKGROUND OF THE INVENTION 
     Avidin is a homotetrameric glycoprotein isolated from chicken egg-white. Each of the eight-stranded beta-barrel subunit of avidin consists of 128 amino acids and has one ligand-binding site. Avidin, like its bacterial analogue streptavidin from  Streptomyces avidinii , is able to form a tight and specific complex with a water-soluble vitamin, d-biotin (dissociation constant K d ≈10 −15  M) (1,2). This special property of avidin together with its tetrameric nature and high stability have made it one of the most widely exploited protein tools in the life-sciences across a range of biochemical, pharmaceutical and biophysical applications (3,4). 
     The avidin gene family consists of avidin and seven avidin-related genes (AVRs) (5). Although avidin protein is expressed in various tissues (6), the other members of the gene family have not so far been found in the form of proteins in the chicken. In order to study their functional and structural properties, AVR proteins were recently produced by a baculovirus insect cell expression system (7). Genes AVR4 and AVR5 both encode identical protein, called AVR4/5. Avidin and AVR4/5 are about 80% identical in amino acid sequence, and almost all of the residues involved in biotin binding in avidin are conserved in AVR4/5. It was found that recombinant AVR4/5 bound biotin almost as tightly as avidin. Most interestingly, it was shown to be significantly more thermostable, the transition midpoint of heat denaturation T m  being 106.4° C. compared to that of avidin, which T m  is 83.5° C. (8). 
     It has been proposed that protein oligomerization in nature serves to obtain more stable structures (9,10). In addition, stable proteins tend to have only a few intrinsic water clefts in their structures (11,12). Moreover, the role of ionic bonds in establishing the high thermal stability of proteins has been studied by Szilágyi and Závodszky (13), who performed a statistical analysis of high-quality protein structures obtained from mesophilic and thermophilic organisms. They observed a correlation between the number of ion pairs and growth temperature of the organism, and hypothesized that ion pairs have structural importance especially at high temperatures. The ionic bonds found in thermostable proteins have successfully been transferred to their analogues from mesophilic organisms in order to stabilize them (14). The importance of aromatic pairs in thermostable proteins has also been noticed (15), and these pairs have successfully been transferred between proteins to improve the thermal stability (16). 
     SUMMARY OF THE INVENTION 
     The chicken avidin gene family consists of avidin and seven separate avidin related genes (AVRs) 1-7 (SEQ ID NOs:2-8). Avidin protein is a widely used biochemical tool whereas the other family members have only recently been produced and characterized as recombinant proteins. Previously, AVR4/5 has been found to be the most stable biotin-binding protein thus far characterized (T m =106.4° C.). The high-resolution structure of AVR4/5 facilitated comparison of the structural details of avidin and AVR4/5. In the present invention, the information obtained from these comparative studies is used to transfer the stability and functional properties of AVR4/5 to avidin. In the present invention higher thermal stability and also better stability against proteolytic degradation were obtained. There are some interesting properties in AVR proteins and it may be possible to move these to the avidin using analogous strategy. 
     A chimeric avidin protein, ChiAVD, containing a 21 amino acid segment of AVR4/5 was found to be significantly more stable (T m =96.5° C.) than native avidin (T m =83.5° C.), and its biotin-binding properties resembled those of AVR4/5. Optimization of a crucial subunit interface of avidin by an AVR4/5-inspired point mutation of isoleucine 117 to tyrosine (I117Y) significantly increased the thermostability of the avidin mutant (T m =97.5° C.) without compromising its high biotin-binding properties. By combining these two modifications, a hyperthermostable ChiAVD(I117Y) was constructed (T m =111.1° C.). We also studied further the biotin-binding properties of AVR4/5. An increase in the energy barrier between the biotin-bound and unbound state of AVR4/5 was observed when compared to that of avidin. The chimeras thus obtained may find a role in applications utilising extreme conditions, like PCR. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  A) Needleman-Wunsch alignment of chicken avidin (SEQ ID NO:17) and AVR4/5 (SEQ ID NO:18). The identity of the proteins is 77.8%. The area covering beta-sheet 4 and parts of its surrounding loops are boxed as well as the mutated amino acid residue I117. The alignment was made using MALIGN (36). B) Topology diagram of the chimeric protein. The segments taken from avidin are shown in black and the part inserted from AVR4/5 is shown in gray. The boundaries between these segments are highlighted by white arrows. 
         FIG. 2  SDS-PAGE analysis of the purified proteins. A=avidin, Y=AVD(I117Y), C=ChiAVD, CY=ChiAVD(I117Y), 4=AVR4/5(C122S), 4b=AVR4/5(C122S)-b produced in bacteria. The marker proteins with molecular weights of 14.4, 21.5 and 31 kDa are shown in figure. 
         FIG. 3  SDS-PAGE analysis of protein samples incubated with Proteinase K. 
       The arrow shows the location of digested protein forms. The sampled marked with “c” are control samples without protease treatment. A=avidin+ProtK, Y=AVD(I117Y), 4=AVR4/5(C122S), C=ChiAVD, CY=ChiAVD(I117Y), 4b=AVR4/5(C122S)-b. The marker proteins with molecular weights of 14.4, and 21.5 kDa are shown in figure. 
         FIG. 4  Radiobiotin dissociation rate constants measured for the proteins as a function of temperature. The dissociation rate constants determined by global fitting are connected by lines. 
         FIG. 5  Tube representation of the superposition of the avidin (magenta) and AVR4/5 (cyan) in the L3,4 loop region with biotin molecules in the binding site for reference. The side chains of residues 39 to 42 are shown for AVR4/5 and indicate the kink in the loop induced by the Pro-Gly tandem. Although the L3,4 loops in both proteins are of the same size, they adopt an entirely different conformation. The closed conformation of L3,4 in the AVR4/5 is apparent in both the apo and biotin complex forms. The figure was constructed using MIDAS (37). 
         FIG. 6  Reaction thermodynamic coordinates for biotin binding to avidin and AVR4/5(C122S). The activation energy of dissociation obtained from the Eyring equation fitted to the dissociation data was used to calculate the transition state energy (ΔG‡) for the reaction, using the total binding energy values (ΔG) previously determined for binding (8). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The chicken avidin gene family consists of avidin and seven separate avidin related genes (AVRs) 1-7. AVR4/5 has been found to be the most stable biotin-binding protein thus far characterized (T m =106.4° C.). The objective of the present invention was to identify the features that make AVR4/5 so much more stable protein than chicken avidin (8), and to transfer this higher stability to avidin. Another objective of the invention was to explore and compare the biotin-binding properties of avidin, AVR4/5 and the chimeric proteins produced in this invention. A further objective was to determine the importance of the differences in the primary and three-dimensional structures (Eisenberg-Domovich et al., manuscript) of these proteins for their biotin-binding and stability properties (8). To accomplish these objectives, molecular modeling and the solved three-dimensional structures were utilized and the results used to transfer the stabilising elements from AVR4/5 to avidin. On the basis of the sequence comparison made between avidin and AVR4/5, the highly variable segment between the two proteins is located between L3,4 and L4,5 ( FIG. 1 ). Firstly, a chimeric avidin was engineered, in which a β4 and its adjacent loops were replaced by the corresponding region from AVR4/5. The thermal stability of the resultant chimera was clearly higher (T m =96.5° C.) than that of avidin (T m =83.5° C.) but lower than that of AVR4/5 (T m =106.4° C.). 
     In another mutant, a point mutation (I117Y) was introduced into avidin from the AVR4/5 sequence on the basis of the modeling (8) and 3-D X-ray structure analysis (Eisenberg-Domovich et al., manuscript), which was thought to play an important role in the intersubunit interactions and thus contribute to the thermal stability of AVR4/5. This substitution raised the thermal stability of the mutant thus obtained to a level (T m =97.5° C.) comparable to that of the above-described chimera. Furthermore, when the two modifications were combined, a hyperthermostable avidin showing even greater thermal stability (T m =111.1° C.) than AVR4/5 was achieved. 
     As expected, a significantly more stable protein was obtained when a 21-long amino acid segment (residues 38-58) taken from AVR4/5 was transferred to avidin by substituting the residues 38-60 of the avidin molecule. The shortening of the L4,5 loop by two residues in the chimeric avidin (ChiAVD) may provide a partial explanation for the higher stability. By comparing genomes of mesophiles, thermophiles and extremophiles, Thompson and Eisenberg found shorter exposed loops from temperature-resistant proteins when compared to those of their mesophilic analogues (25). The L3,4 loop, however has the same length in avidin and AVR4/5, yet its amino acid composition is entirely different ( FIG. 1 ). The three-dimensional structure of L3,4 clearly shows that it has a different conformation in AVR4/5 than in avidin ( FIG. 5 ) (Eisenberg-Domovich et al., manuscript). It is assumed that ChiAVD and AVR4/5 share a similar L3,4 loop conformation. The presence of the Pro41-Gly42 stretch and the salt bridge between Asp39 and Arg112 induces stability in the L3,4 loop in both the apo and biotin complex forms (Eisenberg-Domovich et al., manuscript). 
     The importance of certain stability “hot-spot” residues in protein interfaces has been noticed (26-28). Aromatic pairs are known to form stabilizing pairs in protein structures, which have also been studied experimentally (16). In the present invention, the subunit interface of avidin was optimised by replacing Ile117 residue with tyrosine according to the AVR4/5 sequence. The previous modeling analysis (8) suggested that tyrosine in this location is able to improve the stability of the AVR4/5 tetramer as compared to that of avidin. The three-dimensional structure of AVR4/5 indicates the presence of π-π (pii-pii) stacking between two tyrosine residues from neighbouring monomers (Eisenberg-Domovich et al., manuscript) and experimental data (AVD(I117Y), T m =97.5° C.; avidin, T m =83.5° C.) support the improved structure at this site. Kannan and Vishveshwara compared the aromatic clusters in proteins from thermophilic and mesophilic organisms (15). They found that residues comprising aromatic clusters in proteins from thermophilics are preferably replaced by Leu or lie in proteins from mesophilic organisms. 
     It should be noted that more than one point mutation could be introduced to the protein. In an earlier application FI 20031663, which is incorporated here as reference, new thermally stabilized biotin binding proteins were constructed using site-directed mutation. For avidin and AVRs this was achieved by introducing disulphide bridges between its subunits. 
     From the stability point of view the most interesting result was that combination of the chimera approach and the I117Y point mutation produced a protein that was even more thermostable than AVR4/5. This indicates that the structural factors that account for the difference in stability between avidin and AVR4/5 have successfully been recognized and transferred. It has been proposed earlier that recombination inside the avidin gene family is a frequent event (29). The results in the present application indicate, that recombination may produce functional chimeric proteins inside the gene family, since building blocks moved from AVR4/5 seem to be able to function as part of the avidin structure without negative implications. 
     The enhancement of the stability in ChiAVD is due to substitution of region between beta strands 3 and 5 from AVR4/5. The other AVRs have fairly similar sequence in this region with AVR4/5 and the movement of any of these regions to avidin may allow stabilisation of the end-product similarly as in the case of ChiAVD. 
     For example, avidin has an isoelectric point at high pH i.e it is basic protein. Instead, AVR2 is acidic protein having pl close to pH 5. Therefore, one may be able to change the pl of avidin by moving parts of avidin related proteins to avidin. Simultaneously one could obtain better stability of the end product when compared to wild type avidin. 
     The isoelectric point of avidin was previously lowered by genetic methods by Marttila et al. (38) and Nardone et al. (39), but only individual amino acids were replaced in these studies. 
     The invention will be further described with reference to the following non-limiting examples. 
     EXAMPLES 
     Example 1 
     Construction, Purification and Sequence Analysis of Chimeric Avidin Mutant 
     In order to study the significance of the differing segment between β3 and β5, a ChiAVD (SEQ ID NO:15) was constructed in which this segment was transferred from AVR4/5 (SEQ ID NO:5/SEQ ID NO:6) to avidin (SEQ ID NO:1). The amino acid residues 38-60 of avidin were substituted by 38-58 amino acid residues from AVR4/5 ( FIG. 1 ). Furthermore, isoleucine 117 in avidin was mutated to tyrosine according to AVR4/5 ( FIG. 1 ). 
     Chimeric ChiAVD protein was produced by three sequential PCR reactions, in which the final product was obtained from partially overlapping megaprimer (17) products. The oligonucleotides Kimera.1 and Kimera.2 were used in the first PCR with AVR4 cDNA (7) (SEQ ID NO:5) as a template. The product was isolated by agarose gel electrophoresis and used as a megaprimer in the following PCR with oligonucleotide AK33. Avidin cDNA was used as a template in the second PCR. The product was again isolated by electrophoresis and used again as a megaprimer in the third PCR with oligonucleotide AK44. Avidin cDNA was used as a template in the third PCR. The product was isolated by electrophoresis. 
     The obtained DNA was digested by BgIII and HindIII and ligated to the BamHI/HindIII-digested pFASTBAC1-plasmid. The final product was confirmed by DNA sequencing. 
     Mutation I117Y was made to pFASTBAC1 plasmid coding for avidin or ChiAVD by QuickChange (Stratagene, La Jolla, Calif., USA) method using oligonucleotides I117Y.1 and I117Y.2. The final product was confirmed by DNA sequencing as ChiAVD(I117Y) (SEQ ID NO:16). 
     The final product was cloned to the pFASTBAC1 vector. Recombinant baculoviruses coding for ChiAVD forms, AVR4/5(C122S) and AVD(1117Y) were generated as instructed by the manufacturer of the Bac-To-Bac™ system (Invitrogen). Proteins were produced in baculovirus-infected Sf9 insect cells, in biotin-free medium as reported earlier (7). Non-glycosylated AVR4/5(C122S)-b was produced using the  E. coli  expression system (18) (Laitinen et al., unpublished) in order to study the influence of the carbohydrate chains on the properties of the protein (Table I). The proteins were then purified by affinity chromatography using 2-iminobiotin agarose, as previously described (19). The protein forms are summarised in Table I. 
     Sequences of oligonucleotides: 
     
       
         
               
               
             
           
               
                   
                 Kimera.1 
               
               
                   
                 GCACCTACATCACAGCCGTAGCGGATAATCCAGGAAA 
               
               
                   
                 (SEQ ID NO:9) 
               
               
                   
                   
               
               
                   
                 Kimera.2 
               
               
                   
                 GAAGCCAAAGGTGGGCTGGCTGGCTCTTTTGTGTTGG 
               
               
                   
                 (SEQ ID NO:10) 
               
               
                   
                   
               
               
                   
                 AK33 
               
               
                   
                 CT GCT aga tct ATG GTG CAC GCA ACC TCC CC 
               
               
                   
                 (SEQ ID NO:11) 
               
               
                   
                   
               
               
                   
                 AK44 
               
               
                   
                 GTTGCAAGCTTTGCGGGGCCATCC 
               
               
                   
                 (SEQ ID NO:12) 
               
               
                   
                   
               
               
                   
                 I117Y.1 
               
               
                   
                 CAGGGTCGGCTACAACATCTTC 
               
               
                   
                 (SEQ ID NO:13) 
               
               
                   
                   
               
               
                   
                 I117Y.2 
               
               
                   
                 GAAGATGTTGTAGCCGACCCTG 
               
               
                   
                 (SEQ ID NO:14) 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Description of proteins analysed. 
               
             
          
           
               
                 Protein 
                 Modifications 
                 Source 
               
               
                   
               
               
                 AVD 
                 — 
                 Chicken a   
               
               
                 AVD(I117Y) 
                 Mutation I117Y in 1-3 subunit 
                 BEVS b   
               
               
                   
                 interface. 
               
               
                 ChiAVD 
                 Residues 38-58 moved from AVR4/5 
                 BEVS b   
               
               
                   
                 to avidin. 
               
               
                 ChiAVD(I117Y) 
                 Residues 38-58 moved from AVR4/5 
                 BEVS b   
               
               
                   
                 to avidin and mutation I117Y in 1-3 
               
               
                   
                 subunit interface. 
               
               
                 AVR4/5(C122S) 
                 Cysteine residue forming 
                 BEVS b   
               
               
                   
                 intermonomeric disulphide bridges in 
               
               
                   
                 AVR4/5 mutated to serine. 
               
               
                 AVR4/5(C122S)-b 
                 As above but non-glycosylated. 
                 
                   E. coli 
                   c 
                 
               
               
                   
               
               
                   a Chicken avidin obtained from Belovo S. A. (Bastogne, Belgium). 
               
               
                   b Recombinant protein produced by baculovirus expression vector system in insect cells. 
               
               
                   c Recombinant protein produced by bacterial expression system. This form contains three additional residues (Gln-Thr-Val) in the N-terminus from the bacterial signal peptide (18). 
               
             
          
         
       
     
     Isolated proteins showed high purity in SDS-PAGE analysis ( FIG. 2 ). The glycosylation patterns of the purified proteins differed since AVR4/5(C122S) has three potential glycosylation sites whereas avidin has only one (1,7). One of these sites (Asn43) of AVR4/5 was transferred to the chimeric protein, resulting in more extensive glycosylation of the chimera when compared to that of native avidin. Bacterially produced AVR4/5(C122S) was non-glycosylated as expected ( FIG. 2 ). 
     The sequence identity between avidin and AVR4/5 is 77.8%. More than half (15 of 28 mutations) of the differences between these two proteins are found on the relatively short 23/21 (avidin/AVR4/5) amino acid segment between the end of β3 and the beginning of β5 ( FIG. 1 ). All residues showing contact with biotin (24) are conserved, excluding the Thr38-Ala39-Thr4O-sequence located in the L3,4 loop (connecting β3 and β4 strands), which is replaced by Ala38-Asp39-Asn40 in AVR4/5. Subunit interface residues (41 residues) (8) are also well-conserved, the only amino acid differences being Thr38Ala, Ala39Asp, His50Leu, Thr52Ile, Asn54His and Ile117Tyr (numbering according to avidin sequence). 
     Example 2 
     Proteinase K Assay 
     The proteolytic resistance of the proteins were studied using proteolysis by Proteinase K, as previously described (7). Protein sample (4 μg) was incubated in the presence of Proteinase K ( 1/25 w/w) at 37° C. for a predetermined time period, denatured by boiling in sample buffer (SDS, 2-mercaptoethanol) and subjected to SDS-PAGE followed by coomassie staining. 
     Avidin and avidin mutant AVD(I117Y) were found to be 50% digested after treatment for 16 hours with Proteinase K ( FIG. 3 ). When these proteins were saturated with biotin before treatment, no cleavage was observed. AVR4/5(C122S), however, displayed total resistance to the proteolytic activity of Proteinase K, even without biotin. ChiAVD, ChiAVD(I117Y), as well as AVR4/5(C122S)-b produced in  E. coli , were also found to behave as AVR4/5(C122S) in this assay, i.e. remaining intact for 16 hours in the presence of protease in the presence or absence of biotin. This indicates that the conformation of L3,4 of the AVR4/5 apoprotein (Eisenberg-Domovich et al., manuscript) protects it from digestion. Furthermore, glycosylation in residue Asn43 (8) did not seem to play a role in the protease resistance. 
     Proteinase K cleaves avidin in only one region in the loop between β-strands 3 and 4 (33). It was also found that biotin efficiently inhibits the cleavage. On the other hand, AVR4/5 and streptavidin are resistant to cleavage by Proteinase K, even without bound biotin (8,32). Since Proteinase K cleaves a variety of sequences, the explanation for the resistance to cleavage might lie in the conformation of the L3,4 loop of AVR4/5. The Proteinase K resistance of ChiAVD supports these results ( FIG. 3 ). The different loop structure can be seen in the structure of AVR4/5 ( FIG. 5 ) (Eisenberg-Domovich et al., manuscript). Proline in this loop seems to cause bending in the middle of the loop. Accordingly, the corresponding loop in streptavidin is three residues shorter (34), and may not, therefore, be accessible to the protease. No difference between the glycosylated and non-glycosylated form of AVR4/5(C122S) was observed in this analysis; hence, the sugar moiety in this loop in AVR4/5 cannot explain the resistance to the protease. This is also true for avidin, which showed similar stability in both the enzymatically deglycosylated and normal carbohydrate-containing forms (35). 
     Example 3 
     Gel Filtration Analysis 
     The oligomeric state of the proteins was assayed with FPLC gel filtration as previously described (8). Sodium carbonate buffer (50 mM, pH 11) with 150 mM NaCl was used as the liquid phase. Protein samples of 5-10 μg were used in the analysis. 
     All the engineered proteins showed tetrameric appearance when subjected to gel filtration analysis. Bacterially produced AVR4/5(C122S) showed a slightly lower apparent molecular weight as compared to the other proteins, as expected, owing to the lack of the carbohydrate moiety (Table II). 
     Example 4 
     Microplate Assay 
     The inactivation of the proteins during heat-treatment was analysed by using a microplate assay (23). The proteins were heated to 99.9° C. in 50 mM phosphate buffer containing 100 mM NaCl (pH 7.0) for 32 minutes. The remaining activity was probed by measuring the ability of the proteins to bind biotinylated alkaline phosphatase by coating the microplate wells with samples of the heated proteins. 
     The remaining activity after treatment for 32 minutes is shown in Table II. These results are in line with DSC analyses showing that ChiAVD(I117Y) is the most thermally stable of the proteins characterised. 
     Example 5 
     Differential Scanning Calorimetry (DSC) 
     The transition midpoint of the heat denaturation (T m ) of the avidin proteins was studied using a Calorimetry Sciences Corporation (CSC) Nano II differential scanning calorimeter, as in previous reports (22,23). Proteins (˜0.5 mg/ml) were analyzed both in the absence and presence of biotin (3:1 molar ratio; biotin:avidin monomer). 
     In the DSC-experiments the ChiAVD protein and AVD(I117Y) showed significantly better thermal stability than avidin both as apoforms and holoforms (Table II). When these modifications were combined, the resultant ChiAVD(I117Y) was found to be even more stable than AVR4/5(C122S). In all cases holoforms were clearly more stable than apoforms. We observed a significant increase in the unfolding enthalpy with an increasing melting temperature. AVR4/5(C122S)-b showed similar behavior as compared to the protein produced in insect cells in the DSC analysis. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
             
             
               
                   
               
               
                 Structural properties of avidins. FPLC gel filtration elution times and 
               
               
                 calculated molecular weights of the proteins. Heat-induced unfolding of proteins 
               
               
                 determined by DSC (average ± S.D). 
               
             
          
           
               
                   
                 Gel filtration 
                   
                 Microplate 
               
             
          
           
               
                   
                 Molecular 
                 DSC c   
                 assay 
               
             
          
           
               
                   
                 Elution 
                 mass 
                 T m (−biotin) 
                 T m(+biotin)   
                 ΔTm a   
                 ΔH b   
                 Activity e   
               
               
                 Protein 
                 time (min) 
                 (kDa) 
                 (° C.) 
                 (° C.) 
                 (° C.) 
                 (kJ/mol) 
                 (%) 
               
               
                   
               
             
          
           
               
                 AVD 
                 29.3 
                 53.1 
                 83.5 ± 0.1 
                 117.0 ± 0.7 
                 33.5 
                 329 ± 5  
                 4 
               
               
                 AVD(I117Y) 
                 29.6 
                 49.5 
                 97.5 ± 0.4 
                 123.7 ± 0.1 
                 26.2 
                 536 ± 6  
                 47 
               
               
                 ChiAVD 
                 29.0 
                 56.9 
                 96.5 ± 0.2 
                 124.4 ± 0.2 
                 27.9 
                 527 ± 10 
                 47 
               
               
                 ChiAVD(I117Y) 
                 29.0 
                 56.9 
                 111.1 ± 0.2  
                 ~130 d   
                 ~19 
                 659 ± 38 
                 98 
               
               
                 AVR4/5(C122S) 
                 29.5 
                 50.6 
                 106.4 ± 0.8  
                 125.4 ± 0.8 
                 19.5 
                 575 ± 33 
                 72 
               
               
                 AVR4/5(C122S)-b 
                 30.3 
                 42.1 
                 109.9 
                   127.1 
                 17.2 
                 460 
                 56 
               
               
                   
               
               
                   a ΔT m  is the change in T m  upon addition of a three-fold molar excess of biotin. 
               
               
                   b ΔH value obtained from sample without biotin. The value could not be determined accurately from samples saturated with biotin because the T m  values were too close to the temperature limit of the DSC instrument. 
               
               
                   c The results of AVD and AVR4/5(C122S) are from (8). 
               
               
                   d Could not be determined accurately due to upper limit of temperature of the DSC instrument. 
               
               
                   e Biotin-binding activity after 32 min treatment at 99.9° C. 
               
             
          
         
       
     
     Example 6 
     Optical Biosensor Studies 
     The biotin-binding characteristics of the different avidins were studied by a surface plasmon resonance (SPR) optical biosensor (IAsys). The binding affinities to the 2-iminobiotin surface in 50 mM borate buffer (pH 9.5, 1 M NaCl) were measured as previously reported (7). The result are shown in table III. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE III 
               
             
             
               
                   
               
               
                 2-Iminobiotin-binding properties of proteins determined by IAsys 
               
               
                 optical biosensor. Iminobiotin-binding properties of the proteins 
               
               
                 analysed by an IAsys optical biosensor at 20° C. Affinities to 
               
               
                 2-iminobiotin surface are determined from the equilibrium response 
               
               
                 data. k ass , is an association rate constant. 
               
             
          
           
               
                   
                   
                 K d  (M) a   
                 k ass  (M −1 s −1 ) 
               
               
                   
                   
               
               
                   
                 AVD 
                 (2.1 ± 0.6) × 10 −8   
                 (2.6 ± 0.2) × 10 4   
               
               
                   
                 AVD(I117Y) 
                 (6.3 ± 1.2) × 10 −8   
                 (2.3 ± 0.3) × 10 4   
               
               
                   
                 ChiAVD 
                 (1.1 ± 0.4) × 10 −7   
                 (1.7 ± 0.1) × 10 4   
               
               
                   
                 ChiAVD(I117Y) 
                 (6.7 ± 1.2) × 10 −8   
                 (1.6 ± 0.2) × 10 4   
               
               
                   
                 AVR4/5(C122S) 
                 (1.4 ± 0.4) × 10 −7   
                 (9.3 ± 0.4) × 10 3   
               
               
                   
                   
               
               
                   
                   a Apparent dissociation constant calculated from equilibrium values 
               
             
          
         
       
     
     Example 7 
     Radiobiotin Dissociation Assay 
     The dissociation rate of [3H]biotin from avidin, AVR4/5 and the avidin mutants was determined as described in (20) at various temperatures. The activation thermodynamic parameters for AVR4/5(C122S) and avidin were determined by analysis of the dependence of the dissociation rate upon temperature using the global fit of all data, as described in (21). 
     It was found that AVR4/5 binds biotin almost as tightly as avidin. The analysis of the [3H]biotin dissociation data measured at different temperatures revealed that the energy barrier between unbound and bound states in AVR4 is somewhat smaller than in avidin ( FIG. 6 ). The higher free energy of the transition state might also explain the slower association rate to the 2-iminobiotin surface of AVR4 as compared to avidin ( FIG. 6 , Table III). Because the free energy of the binding is lower in the case of AVR4 (8), the biotin dissociation barrier is still lower for AVR4 despite the higher transition state free energy. 
     A potential explanation for the differences in biotin-binding characteristics between AVR4/5 and avidin lies in the differences in the L3,4 loop. This region has been found to be an important factor in biotin binding to streptavidin (30). Both the ChiAVD forms showed biotin-binding properties similar to those of AVR4/5 when measured by various methods, therefore supporting this hypothesis. We found that glycosylation may play a minor role in biotin-binding, since AVR4/5(C122S)-b produced in bacteria showed slightly tighter binding characteristics in both the radiobiotin and fluorescent biotin dissociation analyses ( FIG. 4 , Table IV). 
     Example 8 
     Fluorescent Biotin Dissociation Assay 
     The binding of labelled biotin to avidins was analysed by a method based on the quenching of a biotin-coupled fluorescent probe ArcDia BF560 (ArcDia Ltd., Turku, Finland) due to binding to avidin, as previously described (18). The measurements were performed using a PerkinElmer LS55 luminometer with thermostated cuvette (25 or 50° C.). The signal measured for 3600 sec (25° C.) or 2400 sec (50° C.) was used to determine the dissociation rate constant. 
     The binding kinetics of the fluorescent biotin conjugate to different avidins were compared by measuring the dissociation rate constants at 25° C. and 50° C. (Table IV). Avidin showed a lower dissociation rate when compared to AVR4/5(C122S). ChiAVD showed characteristics similar to AVR4/5(C122S) in this assay. Interestingly, mutation Ile117Tyr seemed to tighten the binding of the biotin-conjugate to proteins. 
     Ligand-binding analyses done with a IAsys optical biosensor showed a slightly decreased affinity to the 2-iminobiotin surface in the case of ChiAVD as compared to avidin (Table III). However, the affinity was nonetheless high resembling the values found for AVR4/5(C122S) (8). We also observed a slight decrease in the association rate constants of both ChiAVD-forms as compared to avidin and AVD(I117Y). 
     AVR4/5(C122S) showed tight biotin binding in the radiobiotin dissociation assay ( FIG. 4 ). However, the measured dissociation rate constants were significantly higher than those of avidin. ChiAVD and ChiAVD(I117Y) resembled AVR4/5(C122S) in this assay. Interestingly, bacterial AVR4/5(C122S)-b showed a slightly slower dissociation rate in this assay than AVR4/5(C122S) produced in insect cells. Avidin mutant AVD(I117Y) showed very tight, avidin-like biotin binding. The activation thermodynamic parameters were calculated from the data as described elsewhere (21) and combined with the thermodynamic parameters obtained for AVR4/5(C122S) and avidin in a previous study (8). The values obtained are shown in  FIG. 6 . 
     The dissociation rates observed in the fluorescent biotin assay were significantly faster than those obtained from the radiobiotin analysis, but the proteins nevertheless showed similar relative characteristics (Table IV). 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE IV 
               
             
             
               
                   
               
               
                 Biotin-conjugate dissociation kinetics. Dissociation rate constants 
               
               
                 measured for biotin-BF560-conjugate by a luminometer at 25° C. 
               
               
                 and at 50° C. The total releases of the probe after measurement 
               
               
                 for one hour are also shown. k diss  is a dissociation rate constant. 
               
             
          
           
               
                   
                 k diss   
                   
                 k diss   
                   
               
               
                   
                 (10 −5  s −1 ) 
                 Release 1 h 
                 (10 −4  s −1 ) 
                 Release 1 h 
               
               
                   
                 25° C. 
                 (%) 25° C. 
                 50° C. 
                 (%) 50° C. 
               
               
                   
                   
               
             
          
           
               
                 AVD 
                 2.04 
                 10.7 
                 2.74 
                 71.5 
               
               
                 AVD(I117Y) 
                 0.81 
                 3.0 
                 1.61 
                 56.5 
               
               
                 ChiAVD 
                 1.76 
                 13.1 
                 8.00 
                 93.9 
               
               
                 ChiAVD(I117Y) 
                 1.94 
                 9.5 
                 7.33 
                 94.8 
               
               
                 AVR4/5(C122S) 
                 2.77 
                 12.5 
                 7.88 
                 93.7 
               
               
                 AVR4/5(C122S)-b 
                 2.31 
                 10.9 
                 6.20 
                 88.3 
               
               
                   
               
             
          
         
       
     
     Example 9 
     3-D Structure Analysis 
     The information obtained from the avidin (24) and AVR4/5(C122S) (Eisenberg-Domovich et al., manuscript) X-ray structures suggested, that the introduction of β4 and its adjacent L3,4 and L4,5 loops from AVR4/5 to avidin cause no crucial change in the overall shape of the resulting protein. The different conformation of the L3,4 loop of AVR4/5 ( FIG. 5 ), when compared to that of avidin, should be analogously reflected in the properties of ChiAVD, namely in a lower number of hydrogen bonds to the bound ligand (Eisenberg-Domovich et al., manuscript). Furthermore, the interchanged sequence includes the two-residue deletion in loop L4,5, which has observed not to change the structural properties of the surrounding region of the loop in AVR4/5 (Eisenberg-Domovich et al., manuscript). 
     The invention has been illustrated by examples and embodiments, but it may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the enclosed claims. 
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