Patent Application: US-3060500-A

Abstract:
the present invention describes novel beta - sheet proteins having specific binding properties and catalytic properties and also methods for preparing these proteins .

Description:
preparation of a gamma - crystalline mutant with specific binding to the hormone estradiol the design of novel beta - sheet proteins with antigen - binding properties is shown on the basis of isolating a mutant of the bovine gamma - b - crystalline ( gamma - ii ), which binds specifically to the hormone estradiol . specific alteration of selected amino acid positions of a beta - sheet exposed on the surface produced a novel stable protein with beta - sheet structure and specific binding properties . after selecting the beta - sheet region or amino acids suitable for mutagenesis , a site - specific mutagenesis was carried out at the dna level , and in a phagemid a beta - sheet mutant library was prepared , which makes expression and subsequent selection for novel binding properties of the mutants in the phage display system possible . the isolated mutant ( seq id no : 19 ) was compared to the starting protein gamma - ii - crystalline ( seq id no : 20 ) with respect to its new properties . based on the x - ray structure of gamma - ii - crystalline ( wistow et al ., 1983 ), the n - terminal domain of gamma - ii - crystalline ( genbank ® accession no . m16894 ) was selected for mutagenesis . eight amino acids in all , which form a continuous surface segment , were identified there . the selected amino acids are part of a beta - sheet and should not contribute substantially to preserving the structure . they are amino acid positions which are accessible to the solvent and thus also to possible binding partners . the eight amino acids lys 3 , thr 5 , tyr 7 , cys 16 , glu 18 , ser 20 , arg 37 , and asp 39 of seq id no : 22 comprise an area of approx . 6 . 1 % of the total surface area of the protein . the eight amino acid positions were randomized by site - specific mutagenesis . this makes it possible to produce 2 . 6 × 10 10 different protein species . the region to be mutagenized was obtained at the dna level by assembling individual oligonucleotides . this was followed by cloning into a phagemid constructed for selection in the phage display system . for mutagenesis , 227 bp containing the 5 ′ region of the gamma - crystalline mutants with the eight randomized amino acid positions and also suitable restriction cleavage sites were assembled on a solid phase . 10 individual oligonucleotides in all were used therefor , three of which contained the randomized amino acid positions ( fig1 ). during primer synthesis , the nucleotide mixture nn ( t / g ) was used at the eight positions to be mutagenized , resulting theoretically in 32 different codons at one position ( cf . nord et al ., 1997 ). at the start of the assembling , biotinylated oligonucleotides were attached to streptavidin - loaded magnetic beads ( mbs ) from dynal ( m - 280 ). after several attachment , ligation and polymeration steps , it was possible to amplify the pool of mutagenized regions of gamma - crystalline , assembled on the solid phase , by pcr ( fig2 ). the pcr products of approx . 250 bp in length contained an sfi i cleavage site 5 ′ and a bst e11 cleavage site 3 ′. all oligonucleotides used for assembling were adjusted to a concentration of 100 pmol / μl . first , the primers gclie1b ( seq id no : 1 ) and gclie2p ( seq id no : 11 ) were assembled . for this , 36 μl of washing and binding buffer ( wb buffer : 1 m nacl , 10 mm tris - hcl ph 7 . 5 , 1 mm edta ) were added to in each case 4 μl of the primers and the mixture was incubated at 70 ° c . for 5 min . after assembly of the two primers and further incubation at 70 ° c . for 5 minutes , the primer mixture was slowly cooled to room temperature . 4 μl of the gclie1b / gclie2p primer hybrids were mixed with 56 μl of wb buffer and added to 300 μg of the streptavidin - loaded mbs which had been washed beforehand with washing and binding buffer . incubation at room temperature for 15 minutes was followed by washing the mbs with wb buffer and te buffer ( 10 mm tris - hcl ph 7 . 5 , 1 mm edta ). a primer linker fragment is added to the mbs coupled to the first primer hybrid , which fragment is prepared as follows : 4 μl of primer gclib4p ( seq id no : 13 ) or gcli5p ( seq id no : 14 ) are mixed with 36 μl of 1 × ligation buffer from gibco brl ( 50 mm tris - hcl ph 7 . 6 , 10 mm mgcl 2 , 1 mm atp , 1 mm dtt , 5 % ( w / v ) polyethylene glycol - 8000 ). after incubation at 70 ° c . for 5 minutes , both mixtures are combined , incubated at 70 ° c . for a further 5 min and cooled to room temperature . after adding 12 units of t4 dna ligase ( gibco brl ) and 8 μl of 1 × ligation buffer , the reaction mixture is incubated at room temperature for 1 h . 12 μl of this gclie3p / gclib4p / gcli5p bridging fragment are admixed with 54 μl of 1 × ligation buffer and 6 units of ligase , and the mixture is added to the washed mbs containing the first primer hybrid and incubated at room temperature for 1 h . after the ligation reaction , the mbs are washed twice with te buffer and taken up in 64 μl of 1 × ligation buffer containing 8 μl of ligase . 8 μl of the assembled primer mixture gcli6p ( seq id no : 15 )/ gclib7p ( seq id no : 16 ), which primers have been assembled beforehand in analogy to those of gclib4p / gcli5p , were then added to the mbs . the ligation was again carried out at room temperature for 1 h . after washing the mbs twice in te buffer , 12 μl of the 2 nd bridging fragment gclib8p ( seq id no : 17 )/ gclie9p ( seq id no : 18 )/ gclie10 ( seq id no : 2 ) are added and the mixture is ligated for 1 h . the 2 nd bridging fragment is prepared analogously to the first bridging fragment , gclie9p ( seq id no : 18 ) and gclie10 ( seq id no : 2 ) being assembled first and then ligated with gcli8p in the second step . the mbs with the immobilized primers are then again washed with te buffer . the subsequent dna - polymerase and ligase reaction fills in the gaps in the second strand . the mbs are incubated at 37 ° c . for 30 min in the following buffer mixture : 52 . 5 μl of h 2 o , 6 μl of buffer l from boehringer ( 100 mm tris - hcl ph 7 . 5 , 100 mm mgcl 2 , 10 mm dithioerythritol ), 0 . 5 μl of dntps ( 25 mm of each dntp ) and 1 μl ( 2 units ) of klenow fragment ( boehringer ). washing the mbs twice with te buffer is followed by the ligation reaction at room temperature for 1 h . a 100 μl mixture contains 10 units of ligase . after two washing steps with te buffer , the dna strand non - covalently bound to the mbs is removed by treatment with 40 μl of 0 . 1 m naoh for 30 s , and the mbs are resuspended in 60 μl of te . the pcr for amplifying the library is carried out using the mbs as template . the pcr reaction mixture ( 50 μl ) is prepared as follows : 6 μl of mbs , 5 μl of 10 × pcr reaction buffer from stratagene ( 100 mm kcl , 100 mm ( nh 4 ) 2 so 4 , 200 mm tris - hcl ph 8 . 75 , 20 mm mgso 4 , 1 % triton x - 100 , 1 mg / ml bsa ), 1 μl ( 2 . 5 units ) of pfu dna polymerase ( stratagene ), 0 . 5 μl of dntps ( 25 mm of each dntp ), 0 . 35 μl of gclie1b ( seq id no : 1 ). 0 . 35 μl of gclia11b ( seq id no : 3 ) and 36 . 8 μl of h 2 o . the pcr was carried out in 35 cycles with primer annealing at 550 for 1 min , a polymerase reaction at 72 ° c . for 1 . 5 min , denaturation at 95 ° c . for 1 min and a final polymerase reaction at 72 ° c . for 5 min . starting from phagemid pcantab 5e ( pras kit from pharmacia biotech ), a phagemid derivative for cloning the gamma - ii - crystalline mutant band was thus constructed . the entire 3 ′ region of gamma - ii - crystalline ( c - terminal domain : genbank accession no . m16894 ) and the non - mutagenized 5 ′ region were amplified by means of pcr using plasmid pgii ( mayr et al ., 1994 ) as template and primers gcfornot ( seq id no : 4 ) and gcbacksfibst ( seq id no : 5 : see fig3 , 4 ). the sfi i ( gcbacksfibst ; seq id no : 5 ) and not i ( gcfornot ; seq id no : 4 ) cleavage sites introduced by the primers make insertion of the pcr product into phagemid ( gcfornot ; seq id no : 4 ) pcantab 5e possible . together with the gcbacksfibst ( seq id no : 5 ) primer , a bst eii cleavage site was additionally integrated into the gamma - crystalline gene , which allowed cloning of the mutated gamma - crystalline dna fragments . de novo introduction of the cleavage site does not alter the amino acid sequence in gamma - ii - crystalline . after sequencing , the pcr product was cloned as sfi i / not i fragment into phagemid sfi i / not i cut with pcantab 5e . the phagemid pgckt8 - 3 constructed in this way was the starting point for preparing the gamma - ii - crystalline phage display library . phagemid pgckt 8 - 3 was cut with bst e11 and sfi i restriction enzymes and subjected to phosphatase treatment ( shrimps phosphatase from usb ). after the individual cleavages , the dna was fractionated by gel electrophoresis , and the cleaved vector fractions were excised and isolated from the agarose gels by means of electroelution . any further enzymatic treatment was preceded by phenol / chloroform extraction and precipitation of the dna with glycogen . the dna fragment pool which had been amplified by means of pcr and which contained the mutated region of gamma - ii - crystalline was cleaved with sfi i and bst eii restriction enzymes . a total 440 ng of phagemid and 110 mg of pcr product were used for ligating the pcr products into the prepared pgckt 8 - 3 phagemid . the ligations were carried out with a total 44 units of t4 dna ligase ( gibco brl ) in 20 μl mixtures at 16 ° c . overnight . after inactivating the ligase at 70 ° c . for 20 minutes , the ligation reactions were desalted by drop dialysis for 1 h . in each case 30 μl of electrocompetent e . coli tg 1 cells were transformed with in each case 15 μl of the dialysed ligations . the electrocompetent cells were prepared and transformed as described in the pras - kit manual . the transformed cells were created onto glucose - and ampicillin -( 100 μg / ml ) containing sobag plates ( see pras - kit manual from pharmacia - biotech ) and incubated at 30 ° c . overnight . the gcuc - 1 library prepared contained 250 000 original clones . the clones were washed off with 2 × yt medium ( see pras - kit manual ) containing 1 % glucose and 20 % glycerol , aliquoted and stored at − 80 ° c . the amplification factor of the library was determined to 7 × 10 6 . the proportion of recombinant clones in the gcuc - 1 library was 97 %. sequencing of randomly selected clones revealed that codons were used with the expected variants at the randomized amino acid positions . expression rates of 30 - 60 % were detected in the library by means of western - blot analyses . in control experiments , gamma - ii - crystalline dna was amplified using primers gcfornot ( 5 ′ gagtcattctgcggccgcataaaaatccatcacccgtcttaaa gmcc 3 ′; seq id no : 4 ) and gcbacksfi ( 5 ′ catgccatgactcgcggccc agccggccatggggaagatcactttttacgaggac 3 ′; seq id no : 6 ) and plasmid pgii ( mayr et al ., 1994 ) as template . after cleavage with not i and sfi i restriction endonucleases , the sequenced pcr product was cloned into the sfi i / not i phagemid likewise cut with pcantab 5e . the commercially available phage display system pras from pharmacia - biotech was used for selecting gamma - crystalline mutants for binding properties . in the pcantab 5e ( wild - type gamma - ii - crystalline ) and pgckt 8 - 3 ( gamma - crystalline mutants ) phagemids used , the gamma - crystallines are fused n - terminally to the g3 signal peptide and c - terminally to an e - tag which makes immunological detection of the proteins possible ( fig5 ). depending on the bacterial strain used , the amber stop codon after the e - tag is either recognized ( e . coli hb 2151 ), and cleavage of the signal peptide is followed by secretion or overreading of the e . coli cell . after adding a helper phage , recombinant phages can be formed which expose the gamma - ii - crystalline variants on their surface . optimization of cultivation conditions for the gcuc - 1 library and gamma - ii - crystalline wild - type phages under the cultivation conditions described in the pras manual , it was not possible to detect in any of the recombinant phages the expected fusion proteins ( gamma - ii - crystalline / protein 3 ) by means of westerb - blot analyses . only the addition of reduced glutathione ( gsg ) during phage formation altered the redox state in the periplasm of the bacterial cell and thus provided more favourable conditions for phage assembling . when using the gamma - ii - crystalline clone , it was possible to detect recombinant phages carrying the fusion protein only with the addition of gsh . increasing gsh concentration also increased the proportion of gamma - ii - crystalline phages . the optimal gsh concentration was determined to 8 mm . one reason for poor gamma - crystalline expression on the phage surface in the absence of gsh could be the high proportion of reduced cysteines ( 7 ) in gamma - crystalline . when the particularly unfolded gamma - crystalline enters the periplasm , it could , under the oxidative conditions prevailing there , misfold and form aggregates due to the formation of disulphide bridges . this could also suppress phage assembling . when using proteins with reduced cysteines in the phage display system , it may be possible to improve formation of recombinant phages generally by adding gsh . to screen the gcuc - 1 library , all glass equipment used was sterilized at 220 ° c . for 4 h and plastic material was sterilized with helipur for 1 h . gcuc - 1 library panning was carried out using bsa - beta - estradiol 17 - hemisuccinate ( sigma ) as antigen and microtitre plates ( maxisorp from nunc ) as solid phases . during the 3 rounds of panning , the stringency of the washing steps was increased . for the first cultivation , 100 ml of 2 × yt medium containing 2 % of glucose and ampicillin ( 100 μg / ml ) were inoculated with 50 μl of the gcuc - 1 library . the bacteria grew at 37 ° c . and 300 rpm to an od 600 of 0 . 4 . 800 μl of m13ko7 helper phage ( 1 × 10 11 pfu / ml , gibco brl ) were added to 10 ml of this bacterial culture . this was followed by incubation at 37 ° c . for 30 min without and for a further 30 min with gentle agitation ( 50 rpm ). the bacterial pellet was obtained by centrifugation at room temperature and 1500 rpm ( sorvall ss 34 rotor ) for 20 min and taken up in 100 ml of 2 × yt medium containing 8 mm gsh , 100 μg / ml ampicillin and 50 μg / ml kanamycin . the recombinant phages were produced by overnight culturing at 30 ° c . and 300 rpm . the supernatant containing the recombinant phages was obtained by two centrifugations at 10 800 g for in each case 15 minutes and subsequent filtration ( pore size 0 . 45 μm ). the phages were concentrated by adding 1 / 5 of peg / nacl solution ( 20 % peg - 8000 , 2 . 5 m nacl ) to the supernatant , incubating on ice for one hour and two centrifugations at 4 ° c . and 3 300 g for in each case 30 minutes . the phage pellet obtained was suspended in 4 ml of pbs ph 7 . 2 , and remaining cell components were removed by centrifugation ( 10 min , 11 600 g , room temperature ). for the selection process ( panning ), 1 ml of concentrated phages were mixed with 1 ml of a 6 % strength bsa solution ( 6 % bsa in pbs , ph 7 . 2 ) and incubated at room temperature for 10 min . in each case 100 % of the phages treated in this way were added to the antigen - coated microtitre plate wells prepared as follows . nunc - maxisorp microtitre plates were coated with the antigen bsa - beta - estradiol 17 - hemisuccinate . in each ases 100 μl of antigen solution ( 100 μg / ml in pbs ph 7 . 6 ) were introduced into 10 wells in total . the wells coated at room temperature overnight were washed three times with pbs , ph 7 . 6 . free binding sites were saturated by filling the wells with a 3 % strength bsa / pbs solution , ph 7 . 2 , at room temperature for 2 h . prior to adding the bsa - treated phages , the wells were washed twice with a pbs solution ( ph 7 . 2 ). panning was carried out by agitating the microtitre plate gently ( 20 rpm ) for 30 minutes followed by incubation without shaking at room temperature for 90 minutes . unspecifically bound phages were removed by washing 10 times with pbs , ph 7 . 2 / 0 . 1 % tween - 20 and washing 10 times with pbs , ph 7 . 2 . bound phages were eluted by adding in each case 100 μl of 100 mm triethylamine ( freshly prepared ) per well and incubating at room temperature for 10 minutes . the base - eluted phages ( 1 ml ) were neutralized by adding 500 μl of 1 m tris - hcl ph 7 . 4 . 750 μl of these phages were used for infecting 9 ml of tg - 1 cells cultivated on minimal medium plates and having an od 600 of 0 . 4 - 0 . 5 . for this , the bacteria were incubated with the phages at 37 ° c . for 30 min . it was possible to save phages which had bound particularly tightly and had not been removed from the microtitre plate by triethylamine treatment by direct infection of tg - 1 cells . for this , in each case 100 μl of the cultivated tg - 1 cells were added to the wells . after incubating at 37 ° c . for 30 minutes , the infected tg - 1 cells were removed and combined with those from infection with the eluted phages . the infected bacteria were created onto 16 × 16 cm sobag plates and incubated at 30 ° c . overnight . in each case 1 μl of concentrated and eluted phages was used for determining the titre . the bacterial clones obtained were washed off the sobag plates with 12 . 5 ml of 2 × yt , 20 % glycerol . the second and third pannings were carried out similarly to the first with the following changes . phage cultivation was repeated using 20 μl of the washed - off library in 20 ml of medium . 2 ml of the cultivated bacterial culture were used for infection with the helper phage ( bacterial / phages weight ratio : 1 / 20 ). in the second panning the microtitre plates were washed first 15 times with pbs / tween - 20 and then 10 times with pbs and in the 3rd panning first 20 times with pbs / tween - 20 and then 10 times with pbs . concentration of the phages specifically binding to the antigen was detected using a polyclonal phage elisa . in addition to the eluted phages , phages of the staring library gcuc - 1 and of wild - type gamma - ii - crystalline were assayed for comparison . nunc - maxisorp plates were coated with 100 μl of bsa - estradioll7 - hemisuccinate or bsa at a concentration of 2 μg / ml of pbs ph 7 . 6 at room temperature overnight . 3 washings of the wells with pbs , ph 7 . 6 were followed by blocking with 3 % dried milk powder ( glücksklee )/ pbs , ph 7 . 2 at 37 ° c . for 2 h and another ( 3 ) washings with pbs , ph 7 . 6 . the non - concentrated recombinant phages isolated after phage cultivation were firstly blocked at room temperature for 1 h ( 1 : 1 mixture with 6 % strength dried milk powder ( marvel )/ pbs ph 7 . 6 . 100 μl of the blocked phages were applied per well and incubated at 37 ° c . for 1 h . washing the wells in each case 3 times with pbs / tween - 20 and pbs was followed by incubation with the anti - m13 antibody - pod conjugate ( pharmacia - biotech , dilution 1 : 5 000 in 3 % glücksklee / pbs ) at 37 ° c . for 1 h . after washing the plates , the enzyme - bound antibody was detected using 100 μl of immuno - pure - tmb substrate ( pierce ). the colour reaction was stopped by adding 100 μl of 2m h 2 so 4 and extinction at 450 nm was determined . the result of the concentration of the phages binding to the bsa - estradiol conjugate , after the 3rd panning , is shown in fig6 . isolation and characterization of individual phages with specific binding to the conjugate 80 individual clones were selected from the bacterial clones obtained after the 3rd panning . phages were isolated from the clones and assayed individually in the monoclonal phage elisa with respect to their antigen binding . individual bacterial clones were cultivated in 100 μl of 2 × yt medium containing 2 % glucose and 100 μg / ml ampicillin in polypropylene microtitre plates ( nunc ) with gentle agitation ( 100 rpm ) overnight . 2 μl of these bacterial cultures were diluted 1 : 100 in the same medium and cultured at 100 rpm at 37 ° c . to an od 600 of 0 . 4 . phages were obtained as described for the selection process . deep well polypropylene microtitre plates from tecan were used for phage cultivation . for the elisa , 200 μl of the phage supernatant obtained after centrifugation ( not concentrated ) were blocked with 40 μl of 6 × pbs / 18 % at room temperature for 1 h . 30 out of 80 clones assayed showed significant binding of the recombinant phages to bsa - estradiol - 17 and not to bsa assayed in parallel . phages with wild - type gamma - ii - crystalline showed in a control experiment no binding to bsa - estradiol - 17 whatsoever . 14 selected binding phages were sequenced using the ird 800 - labelled primers pcanr1lab ( 5 ′ ccatgattacgccaagctttggagcc 3 ′; seq id no : 7 ) and gcliseq ( 5 ′ ctgaaagtgccggtgtgttgc 3 ′; seq id no : 8 ). only in one case , sequencing revealed a gamma - crystalline variant ( mu 12a ) which was mutated exclusively in the eight randomized amino acid positions . a number of clones showed shifts in the reading frame and , although theoretically coding for a functional protein , had alterations which were not exclusively in the expected gamma - crystalline region . these frame shift mutants were not studied further . expression of the fusion protein mu 12a - minor coat protein 3 on the surface of the recombinant phages and expression of mu 12a in e . coli strain hb 2151 were detected by means of western - blot analyses using the anti - g3p and anti - e - tag antibodies ( pharmacia - biotech ), respectively . the dna sequences of mutant 12a ( seq id no : 9 ) in phagemid pgckt 8 - 3 and of gamma - ii - crystalline wild - type ( seq id no : 10 ) are depicted in fig7 . the dna sequence starts at the sfi i cleavage site which is already present in the starting phageimid pcantab 5e and ends , in the case of pgckt 8 - 3 , at the bst e11 site newly introduced into the gamma - ii - crystalline gene and , in the case of the gamma - ii - crystalline wild - type gene , at the original sequence . fig8 depicts the amino acid sequences derived therefrom . codon randomization at amino acid position 36 does not change the amino acid arginine at this position . computer modeling of mutant 12a ( seq id no : 19 ) shows that the amino acid exchanges do not cause large alterations in the protein structure compared with the starting protein . however , the net charge becomes more positive . in order to characterize mutant 12a in detail , the dna ( seq id no : 9 ) was recloned into plasmid pet - 20b ( novagen ). the plasmid makes possible a high expression of the recombinant dna in e . coli strain bl 21 and simple purification of the foreign proteins . genes are expressed here without signal peptide and with a c - terminal fusion of 6 histidine residues . the dnas of mutant 12a ( seq id no : 9 ) and of bovine gamma - ii - crystalline wild - type ( seq id no : 10 ) were amplified by means of pcr using the appropriate phagemid dna and primers gc 20bback12a / gc ( seq id no : 24 ) for 20b for mutant 12a and gc 20bbackwt / gc ( seq id no : 23 ) for 20b for the wild - type ( fig9 ). the pcr fragments were cleaved with restriction endonucleases nde i and bam hi and cloned into vector pet20b cut with nde i / bam hi . fig1 depicts the theoretical amino acid sequence of mutant 12 a ( seq id no : 21 ) and of gamma - ii - crystalline ( seq id no : 22 ), respectively , after expression in pet - 20b . the first 10 n - terminal amino acids of mutant 12 a ( seq id no : 21 ) were confirmed by n - terminal protein sequencing . in order to study the binding properties and stability of the mutant in detail , large amounts of mutant 12a and wild - type proteins were prepared . bl 21 cells were transformed with plasmids pet - 20b / mu 12a and pet - 20b / gamma - ii - crystalline , respectively . the clones were cultivated by diluting a preculture 1 : 100 with lb medium / 100 μg / ml ampicillin and agitating the culture at 200 rpm and 37 ° c . up to an od 600 of 0 . 5 . expression of the gamma - crystalline was induced by adding iptg ( final concentration 1 mm ). culturing was continued overnight at 30 ° c . and 200 rpm . the bacteria cells were harvested by centrifugation at 4 ° c ., 6 000 rpm ( sorvall gs3 rotor ) for 10 min . the cell pellet was suspended in 30 ml of 2 × pbs with addition of 150 μl of 200 mm pmsf and 10 μl of dnase ( boehringer ). the cells were then disrupted twice using a gaulin press at 800 - 1000 psig . the supernatant containing the soluble proteins was obtained after centrifugation of the cell suspension at 4 ° c . and 20 000 rpm ( sorvall ss 34 rotor ) for 1 h . the gamma - crystallines fused to 6 histidine residues were purified by affinity chromatography at 4 ° c . 8 ml of ni - nta were equilibrated with 50 ml of 2 × pbs / 10 mm imidazole . the supernatant containing the soluble proteins was then slowly agitated with the equilibrated column material in a bach process on a roller shaker overnight . introducing the suspension into a chromatography column was followed by washing with 2 × pbs / 10 mm imidazole / 300 mm nacl . the bound protein was eluted with 2 × pbs / 250 mm imidazole . dtt ( final concentration 10 mm ) was added to the eluted proteins . this was followed by 2 dialysis steps at 4 ° c . for in each case 8 h : 1st with 100 mm na phosphate buffer ph 6 . 0 / 1 mm edta / 1 mm dtt and 2nd with 10 mm na phosphate buffer ph 6 . 0 / 1 mm edta . the supernatant obtained after a final centrifugation ( 4 ° c ., 30 min , 20 000 rpm in sorvall ss 34 rotor ) contained the purified protein ( mu 12a or gamma - ii - crystalline ) which was used for binding studies and stability studies . specific binding of mutant 12a to the bsa - estradiol - 17 - hemisuccinate conjugate was assayed by carrying out an elisa , with increasing concentrations of purified mutant 12a - his - tag protein being used . increasing amounts of gamma - ii - crystalline wild - type ( likewise with his - tag ) were used as control , and binding of both purified proteins to bsa was assayed . the concentration - dependant elisa was carried out using nunc - tm plates . antigen coating with the bsa - estradiol - 17 - hemisuccinate conjugate or with bsa was carried out at room temperature overnight . coating was carried out with in each case 100 μl of antigen at a concentration of 20 μg / ml of pbs ph 7 . 6 . after washing ( 2 × pbs ph 7 . 6 ) and blocking the plates ( 3 % marvel / pbs at 37 ° c . for 2 h ), in each case 1 - 13 μl of the protein stock solution ( concentration 0 . 63 mg / ml ) of purified mu 12a or gamma - ii - crystalline were introduced into a total 100 μl of reaction solution ( pbs , 3 % marvel , x μl of protein ) and incubated in the wells at 37 ° c . for 2 h . the secondary antibodies used were the tetra - his antibody from qiagen in a dilution of 1 : 3000 and the anti - mouse pod antibody ( sigma ) in a dilution of 1 : 2000 . the antibodies were diluted with a 3 % strength marvel / pbs solution and 100 μl were added to the wells and incubated at 37 ° c . for in each case 1 h . the substrate reaction was carried out as described for the polyclonal phage elisa . the result of this elisa in fig1 shows clearly that increasing extinctions are measured only with increasing concentrations of mutant 12a . no increase was detected using gamma - ii - crystalline . likewise , no reaction with bsa was observed . this shows specific binding of mutant 12a compared with the starting protein . stability was studied by recording guanidine denaturation because of mutant 12a and of gamma - ii - crystalline . for this purpose , the purified proteins were incubated at a final concentration of 20 μg / ml with increasing concentrations of guanidinium at 20 ° c . for one and three days . in total 15 guanidinium concentrations were adjusted in a range from 0 - 5 . 5 m in a 1 mm dtt / 0 . 1 m na phosphate buffer ph 6 . 0 solution . after one and three days , respectively , a 300 - 400 nm fluorescence emission spectrum of each mixture was recorded . the excitation wavelength was 280 nm . fig1 depicts the emission maxima determined as a function of guanidinium concentrations . the stability of gamma - ii - crystalline is higher than that of mutant 12a both after one day and after three days . however , compared with antibody molecules , the stability of mutant 12a is much higher . fluorescence spectra were recorded in order to test whether the fluorescence properties of mutant 12a have changed compared with wild - type protein . for this purpose , in each case 100 μg / ml of wild - type protein or mutant 12a ( in 50 mm na phosphate , ph 6 . 0 ) were excited at 280 nm and fluorescence was measured in a wavelength range from 300 to 400 nm in a cuvette of 1 cm pathlength . the slit width was 5 nm both for excitation and for emission . the detected fluorescence signal had a maximum of 329 nm both for wild - type and for mutant 12a . however , the fluorescence intensity of mutant 12a , with only 86 % signal intensity , was distinctly lower compared with gamma - crystalline wild - type ( 100 %) ( see fig1 a ). mutant 12a and wild - type have an identical total number of fluorophores . however , sequence alterations in the mutant ( y → k at position 8 and c → y at position 15 ) cause a change in the fluorescence signal . the difference in fluorescence intensity can be attributed to the fact that the tyrosine residues in positions 8 and 15 , respectively , have different fluorescence properties . bird , r . e ., hardman , k . d ., jacobson , j . w ., johnson , s ., kaufman , b . m ., lee , s .- m ., lee , t ., pope , h . s ., riordan , g . s , and whitlow , m . 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