Patent Application: US-28333205-A

Abstract:
the invention relates to modified proteins of the superfamily of “ ubiguitin - like proteins ”, proteins that have a ubiguitin - like fold and fragments or fusion proteins thereof . as a result of said modification , the proteins have a binding affinity with respect to a predetermined binding partner that did not exist previously . the invention also relates to a method for the production and utilization of said proteins .

Description:
fig1 : the crystallographic structure of the wild - type ubiquitin ( pdb code : lubi ; vijay - kumar et al ., 1987 ) with the artificially generated binding site on the surface . the three - dimensional representation of the secondary structural elements was performed using the pymol program ( delano scientific , san francisco ). the residues to be randomly substituted in the library prepared as an example comprise positions 2 , 4 , 6 , 62 , 63 , 64 , 65 , 66 , and are shown with their side chains . fig2 : generation of a binding site on the surface of wild - type ubiquitin ( pdb - code : lubi ). the residues to be randomly substituted in the library to be prepared ( positions 2 , 4 , 6 , 62 , 63 , 64 , 65 , 66 represented in light grey ) were selected by computerized analysis with respect to their exposition to the solvent and the stabilizing or destabilizing effect , respectively , which an amino acid substitution could have at the respective positions . using the prosaii software the protein stability of each of 10 4 variations was then determined in comparison to ubiquitin ( wt ) and the same number of randomly taken samples of variations in which the residues of a “ control epitope ” ( positions 24 , 28 , 31 , 32 , 35 , 37 , 38 , 39 represented in dark grey ) were substituted . as a measure for stability the value of the combined c α / c β potentials (“ zp - comb ”) is noted . approximately 19 % of the variations generated in silico which had been randomly substituted in the region of the binding site had a stability which was at least as high as that of ubiquitin ( wt ) while about 90 % were more stable than those bearing the “ control epitope ”. the generation of a binding site by random amino acid substitutions in the region analyzed should therefore be tolerated by the protein structure of ubiquitin without drastically affecting the stability thereof . this computerized method assists in the selection of suitable surface - exposed amino acids . fig3 : the phasmid vector pmubi - 1 for use in the selection of mubi variations by phage display . pmubi - 1 is used for the preparation of phage libraries and codes for a fusion protein of the pelb signal sequence , the gene for the ubiquitin variation , a mycut tag and a c - terminal fragment of the phage capsid protein ( aa 253 - 406 , delta gpiii ) under the transcriptional control of the tetracyclin promoter / operator ( tet p / o ). amber refers to the position of the amber stop codon in the fusion gene . the insertion of the mutated gene cassette of mubi is carried out via the two sfii restriction sites . f1ig , bla p , tetr , ori , cole1 and cat refer to the intergenic region of phage f1 , the tetracyclin repressor gene under the control of the β - lactamase promoter , the origin of replication and the chloramphenicol acetyltransferase gene . fig4 : procedure for the construction of a phage display library of ubiquitin variations . fig5 : binding of the ubiqitin variation spu - 1 - d10 obtained from the selection by means of phage display against recglp1 - r to the aminoterminal domain of the human glp - 1 receptor prepared by recombination in the elisa . an appropriate number of wells of a microtiter plate was filled over night at 4 ° c . with recglp1 - r and with bsa , respectively , or the aminoterminal domain of the human pth receptor ( recpth - r ) prepared by recombination . remaining binding sites were saturated by incubation with 3 % bsa in pbs containing 0 . 5 % tween ( 2 hrs ., room temperature ( rt )). spu - 1 - d10 was applied in serial dilutions in pbst 0 . 1 for 90 min at 30 ° c . bound modified protein was detected with ubiquitin antiserum ( 1 : 10 in pbst 0 . 1 , 60 min at 30 ° c .) and rabbit antibodies ( 1 : 2000 in pbst 0 . 1 , 60 min at 30 ° c .) conjugated to peroxidase . the detection was carried out using the immunopure kit of pierce . between the steps washing was carried out was three times with pbst 0 . 1 — an additional 3 × with pbs prior to chromogenic detection . the signal intensity was detected at 405 nm after stopping the color reaction with h 2 so 4 and plotted against the corresponding concentration of the modified protein . determination of the k d value under equilibrium conditions was carried out by non - linear regression . fig6 : binding of the ubiquitin variation spw - 11 - a1 obtained from the selection by means of ribosomal display on vegf in the elisa . the same procedure as described in fig5 was followed in this experiment . determination of the k d value under equilibrium conditions was carried out by non - linear regression . fig7 : binding of the ubiquitin variation spu - 11 - 58 obtained from the selection by means of ribosomal display on vegf in the biacore . on the surface of the channel of a cm5 chip first vegf was immobilized while a reference channel remained uncharged . for the application of different concentrations of spu - 11 - 58 the net signal of the binding event was obtained in each case . the injection period for all solutions was 2 min at a flow rate of 35 μl / min . the binding signals of different concentrations of spu - 11 - 58 ( 1 . 3 μm , 0 . 65 μm , 0 . 325 μm , 0 . 163 μm , 0 . 081 μm ) are shown superimposed in relation to the start of injection . after every measurement the surface was regenerated at a pulse of 15 s with 10 mm hcl . determination of the k d value under equilibrium conditions was carried out using the biaevaluation software 3 . 1 ( biacore ). fig8 : binding of the ubiquitin variation spu - 3 - h13 obtained from the selection against hydrocortisone to steroids in the elisa . three wells each of a microtiter plate were filled with a bsa conjugate of hydrocortisone , testosterone and oestradiol or with bsa , respectively , and incubated with spu - 3 - h13 ( 4 μm ) or with the ubiquitin protein scaffold which was unchanged in the region of the binding site ( 50 μm ), respectively , each in pbst 0 . 1 ( 90 min at 30 ° c .). the detection of binding is carried out with ni — nta / peroxidase conjugate ( 1 : 500 in pbst 0 . 1 , 60 min at 30 ° c .) and using the immunopure kit of pierce . blocking and washing : see fig5 . the signal intensity was detected at 405 nm after stopping the color reaction with h 2 so 4 and the mean values of three measurements were plotted . fig9 : comparison of the binding of the ubiquitin variations spu - 2 - a7 and spu - 2a7 ( 62 / 63 ) to f c igm following affinity maturation by means of site - directed random mutagenesis in the elisa . the same experimental procedure as in fig5 was followed . determination of the k d value under equilibrium conditions was carried out by non - linear regression . fig1 : analysis of the site - directed , covalent coupling of two ubiquitin - based proteins via single , carboxyterminal cysteine residues by sds - page . 20 μl each of the uncoupled protein ( lane 1 ) as well as the sample after the coupling reaction ( lane 2 ) were applied . following electrophoresis the gel was stained with coomassie . the molecular weights of the size standard ( lane m ) are given in kda . provision of a synthetic ubiquitin gene for the selection of modified proteins having a newly generated binding affinity genetic engineering work was performed by standard protocols known to those skilled in the art such as e . g . those of sambrook et al . ( 2001 ). for the preparation of the dna sequence ( seq id no . 1 ) for a modified ubiquitin protein scaffold having the substitutions ile44ala , lys48arg , arg54leu , val70ala , arg72leu , gly75ala as well as the deletion of gly76 as a starting point for the preparation of artificial binding proteins the procedure was as follows : for gene synthesis a pcr reaction was performed in a volume of 50 μl in which 2 . 5 μl each of the six oligodeoxynucleotides ( seq id no . 2 , seq id no . 3 , seq id no . 4 , seq id no . 5 , seq id no . 6 , seq id no . 7 ; 0 . 1 μm each ) representing together in their base pair sequence the gene to be synthesized were present as templates . the sequences of the oligodeoxynucleotides employed each corresponded to segments of the coding and the non - coding dna strand , respectively , of the artificial gene with a length of 40 to 50 base pairs alternatingly overlapping at their 3 ′ and 5 ′ ends by approx . 15 bases . in addition , the sample contained 2 . 5 μl each of flanking primers ( seq id no . 8 , seq id no . 9 ; 10 μm ) as well as 5 μl of 10 × taq buffer ( 100 mm tris / hcl ph 9 . 0 , 500 mm kcl , 1 % ( v / v ) triton x - 100 ), 3 μl 25 mm mgcl 2 , and 4 μl dntp mix ( 2 . 5 mm each of datp , dctp , dgtp , dttp ). after filling up with h 2 o the reaction sample was heated in the thermocycler for denaturation for 2 min to 94 ° c . then , 2 . 5 u taq polymerase ( promega ) were added during heating ( hot start ) and the pcr program was started . incubation was performed for 25 cycles of each 1 min at 94 ° c ., 1 min at 55 ° c ., and for 1 . 5 min at 72 ° c . a final incubation was carried out for 5 min at 72 ° c . the desired pcr product was identified by means of analytical agarose gel electrophoresis and purified from the sample using the minelute reaction cleanup kit ( qiagen ). 1 . 0 ng of the isolated dna were used as a template for a second amplification , this time using pfu polymerase ( promega ) also in a volume of 50 μl . for this purpose , 5 μl of the supplied 10 × pfu buffer ( 200 mm tris / hcl , ph 8 . 8 , 20 mm mgcl 2 , 100 mm kcl , 100 mm ( nh 4 ) 2 so 4 , 1 % ( v / v ) triton x - 100 , 1 mg / ml bsa ) as well as 4 μl dntp mix were used and filled up with h 2 o . in addition , the sample contained flanking primers ( seq id no . 8 , seq id no . 9 ; 10 μm ) for the introduction of suitable restriction sites . the desired pcr product was isolated by means of preparative agarose gel electrophoresis and was inserted into the cloning vector pcr ® 4blunt - topo ® using the zero blunt ® topo ® pcr cloning kit ( invitrogen ) according to the manufacturer &# 39 ; s instructions . the chemically competent cells supplied were transformed with the corresponding ligation reaction sample and spread on an agar plate in lb / amp / kan medium . the plate was incubated for 16 hrs . at 37 ° c ., and the colonies grown were analysed for the desired ligation product . for this purpose , plasmid dna was prepared in the mini scale using the plasmid isolation kit of quiagen company according to the manufacturer &# 39 ; s instructions , and was subjected to a restriction digest with the dna endonucleases ndei and xhoi ( new england biolabs ) for which the recognition sequences had been introduced into the pcr product by the flanking primers . a sequence analysis was performed on plasmids having the expected cleavage pattern in the region of the gene cassette inserted using taq dna polymerase . for this purpose , the cyclereader ™ autodna sequencing kit ( fermentas ) was used according to the manufacturer &# 39 ; s instructions as well as 0 . 5 μg of plasmide dna and 1 . 0 pmoles of the respective fluorescence - labeled primer . the newly synthesized dna strand was labeled during the polymerase reaction and terminated statistically , but in a base - specific manner by the incorporation of dideoxynucleotides . the fluorescent dna fragments obtained were then separated in a liquor sequencing apparatus by polyacrylamide - urea gel electrophoresis and visualized as a band pattern for a , c , g , t in adjacent lanes . gene cassettes having the correct dna sequence were cut from the cloning vector pcr ® 4blunt - topo ® by preparative ndei / xhoi restriction digest and isolated by preparative agarose gel electrophoresis . the insertion of the gene for the modified ubiquitin protein scaffold is carried out into the expression vector pet20b (−) ( novagen ) for the production of the corresponding protein or into the phasmid vector pmubi - 1 for the construction of a library of ubiquitin - variations , respectively . for random site - specific mutagenesis of 8 codons at the amino and carboxy terminus , respectively , of the synthetic ubiquitin gene two successive pcr reactions were performed . the first amplification step was performed using pfu polymerase ( promega ) in a volume of 10 × 50 μl . for this purpose , 5 μl of the supplied 10 × pfu buffer as well as 4 μl dntp mix were used per each sample and filled up with h 2 o . furthermore , each sample contained 2 . 5 μl of flanking primers ( seq id no . 10 , seq id no . 11 ; 10 μm ) for the introduction of the desired base pair substitutions . as a template , 1 . 0 ng pmubi - 1 were used carrying the non - mutated synthetic ubiquitin gene . following the addition of 2 . 5 u of pfu polymerase ( see above ) an incubation was performed for 25 cycles of each 1 min at 94 ° c ., 1 min at 60 ° c . and for 1 . 5 min at 72 ° c . a final incubation was carried out for 5 min at 72 ° c . for the selective degradation of the template dna employed 10 u dpni were added per reaction sample and incubated for 1 hour at 37 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). the second amplification step was performed in a sample volume of 1 , 000 μl wherein approx . 1 . 0 ng of the product obtained in the first pcr reaction were used and taq polymerase was employed . the reaction sample was pipetted — adapted to 20 times the volume — as detailed above consisting of 10 × taq buffer , 25 mm mgcl 2 , dntp mix as well as the flanking primers ( seq id no . 12 , seq id no . 13 ; 10 μm ) which were biotinylated at their 5 ′ ends and each carrying recognition sequences for sfii endonuclease which were not compatible with each other . after filling up with h 2 o , 2 . 5 u of taq polymerase were added in the heat ( see above ) and the pcr program was started . an incubation was performed for 25 cycles of each 1 min at 94 ° c ., 1 min at 60 ° c . and for 1 . 5 min at 72 ° c . a final incubation was carried out for 5 min at 72 ° c . the subsequent cleavage of the amplification product obtained is carried out directly in the pcr reaction sample . for this purpose , in total volume of 4 , 000 μl the complete pcr reaction solution was mixed with the corresponding volume of the supplied 10 × buffer ii ( 100 mm tris / hcl , ph 7 . 9 , 100 mgcl 2 , 500 mm nacl , 10 mm dithiothreitol ), 10 × bsa solution and h 2 o . furthermore , 4 , 000 u of the restriction enzyme sfii ( new england biolabs ) were added and incubated for 16 hrs . at 50 ° c . the dna was isolated from the sample using the minelute reaction cleanup kit ( qiagen ) and resuspended in 400 μl of sterile h 2 o . for the separation of the pcr product which was not cleaved by sfii the isolated dna was mixed with the same volume of “ binding solution ” ( dynal ) containing 1 . 0 mg / ml magnetic beads having streptavidine coupled to their surface (“ dynabeads kilobase binder ”) and incubated for 4 . 5 hrs . on a roller mixer at room temperature ( rt ). the beads with the optionally still present biotinylated dna were precipitated while dna completely cleaved by sfii which should no longer have biotinylated ends remained in the supernatant and was precipitated over night . the ubiquitin gene mutagenized at the desired positions and cleaved by sfii obtained in this manner was dissolved in sterile h 2 o , again desalted using the qiaquick pcr purification kit ( qiagen ) and finally had a concentration of 200 fmol / μl in h 2 o . for the preparation of the recipient vector the phasmid pmubi - 1 was cut with sfii according to the manufacturer &# 39 ; s instructions and the larger ( vector -) fragment was isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). to avoid intramolecular ligation the 5 ′ ends thereof were dephosphorylated . for this purpose , 0 . 5 u of alkaline phosphatase from shrimp ( pandalus borealis ) as well as the buffer supplied were used in a total volume of 200 μl . the mixture was incubated for 90 min at 37 ° c ., the dna isolated from the sample using the qiaquick pcr purification kit ( qiagen ) and again desalted ( qiaquick pcr purification kit ). the dna of the vector fragment finally had a concentration of 50 fmol / μl in h 2 o . for ligation 1 . 6 pmol of the pcr fragment and 8 . 0 pmol of the vector fragment of pmubi - 1 were incubated in the presence of 2 u t4 dna ligase ( gibcobrl ) in a total volume of 1 , 600 μl ( 50 mm tris / hcl , ph 7 . 6 , 10 mm mgcl 2 , 1 mm atp , 1 mm dtt , 5 % ( w / v ) peg - 8 , 000 ) for three days at 16 ° c . after heating the sample to 65 ° c . for 15 min the dna was precipitated . for this purpose , 100 μl of each of the reaction solutions were mixed with 100 μl ethanol as well as 10 μl 5 m naac , ph 3 . 0 and kept for 16 hrs . at − 20 ° c . subsequently , a centrifugation was carried out ( 60 min , 12 , 500 g ), the sample was washed with ethanol ( 70 % v / v , − 20 ° c . ), recentrifugated , and the precipitated dna was finally dissolved in 60 μl of sterile h 2 o . for electroporation the gene pulser ® ii system ( biorad ) as well as cuvettes having an electrode spacing of 1 . 0 mm ( biozym ) were used at 4 ° c . in the cold room . using 3 . 5 μl of each of the solutions obtained above electrocompetent e . coli xl1blue ( stratagene ) were transformed according to the manufacturer &# 39 ; s instructions . the cell suspension obtained was plated onto five agar plates ( 20 × 20 cm ) with lb / chloramphenicol medium . the plates were incubated for 16 hrs . at 37 ° c . and the colonies grown were counted . the library constructed was found to include 2 . 8 × 10 7 independent clones each of which should be present 10 , 000 times in the library . then , the colonies were floated off in a total of 100 ml soc medium containing 10 % ( v / v ) glycerol and stored in aliquots of 1 . 0 ml at − 80 ° c . the phasmid vector was isolated from 12 clones which were randomly selected among those obtained using the dna miniprep kit of qiagen company and the dna sequence was analysed in the region of the mutagenized ubiquitin gene . all of these clones had functional sequences — i . e . no reading frame shifts by insertions or deletions — as well as qualitatively completely different substitutions at the mutagenized positions . random substitutions outside of the mutagenized regions were not present . preparation of ubiquitin variations on the phage surface and selection of ubiquitin variations against proteins and haptens for the production of phagemids variations of the mutated ubiquitin presented on the surface , 100 ml of 2 × yt / chloramphenicol medium were inoculated with 1 . 0 ml of the glycerol culture obtained in example 2 and incubated for 16 hours at 37 ° c . and 220 ppm . with 10 ml of this stationary culture 1 liter of 2 × yt / chloramphenicol medium was inoculated and agitated at 37 ° c . and 220 rpm up to a cell density of od 600 = 0 . 4 . the infection was carried out with 10 13 cfu of m13ko7 helper phages and an incubation at 37 ° c . without shaking for 30 min . after addition of 50 mg / l kanamycine the sample was agitated for 30 min at 37 ° c . and 220 rpm , and then the gene expression on pmubi - 1 was induced by adjusting the culture to 0 . 2 mg / l anhydrotetracycline ( stock solution : 2 . 0 mg / ml in dmf ). the incubator temperature was then decreased to 26 ° c ., and the culture was agitated for 16 hours at 220 rpm . finally , the cells were sedimented by centrifugation ( 30 min , 12 , 000 g , 4 ° c .) and discarded while the supernatant was filtered ( 0 . 45 μm ). the phagemids contained were precipitated by addition of ¼ volume of 20 % ( w / v ) peg 6 , 000 , 2 . 5 m nacl and incubation for 1 hour on ice and sedimented by centrifugation ( 30 min , 12 , 000 g , 4 ° c .). then , the phagemids were dissolved in 4 ml of ice - cold pbs ( 137 mm nacl , 2 . 7 mm kcl , 8 mm na 2 hpo 4 , 2 mm kh 2 po 4 ), stored for 30 min on ice and centrifuged ( 30 min , 12 , 000 g , 4 ° c .). the supernatant was added with ¼ volume of 20 % ( w / v ) peg 6 , 000 , 2 . 5 m nacl , the phagemids were again precipitated by incubation for 1 hour on ice and precipitated by centrifugation ( 30 min , 12 , 000 g , 4 ° c .). then , the phagemides were again solved in 4 ml ice - cold pbs , stored for 30 min on ice and centrifuged ( 30 min , 12000 g , 4 ° c .). the supernatant was mixed 1 : 1 with 4 % ( w / v ) bovine serum albumin ( bsa ) in pbs , incubated for 30 min on a roller mixer at rt and then directly used in the affinity enrichment . as an affinity matrix for the isolation of phagemids with surface - presented ubiquitin variations which should bind to previously defined target substances , 24 wells each of a microtiter plate were coated with the respective substance . as the target substances served the recombinantly prepared aminoterminal domain of the human glp - 1 receptor ( recglp1 - r ; bazarsuren et al ., 2002 ), the fc portion of human immunoglobulin m ( fc igm ) as well as hydrocortisone ( hc ) coupled to bsa which were immobilized over night at 4 ° c . on the microtiter plate . unoccupied binding sites on the surface of the microtiter plate were blocked by incubation of each of the wells with 400 μl of 4 % bsa in pbs for 90 min at rt . then , 100 μl of the phagemid solution prepared were pipetted into each well and incubated at rt ( room temperature ) for 90 min . unbound phagemids were then removed by washing twice with pbs containing 0 . 05 % ( v / v ) tween 20 ( pbst 0 . 05 ), incubating twice with 400 μl of 4 % bsa in pbs for 5 min and washing twice with pbs as well as vigorous tapping . phagemids bound to the affinity matrix were finally eluted by means of 100 μl of 100 mm triethylamine per well and incubation for 10 min at rt . the solutions containing the eluted phagemids were combined — each separately depending on the respective target substances — and immediately neutralized by addition of ½ volume of 1 m tris / hcl , ph 7 . 4 . the solution obtained in each case was transferred to 20 ml of a culture having a cell density of od 600 = 0 . 4 for the infection of e . coli xl1 blue and agitated for 30 min at 37 ° c . and 220 rpm . the wells of the microtiter plate were again washed ( 5 × pbst , 0 . 05 , 1 × pbs ) and added with 100 μl each of a culture of e . coli xl1 blue having a cell density of od 600 = 0 . 4 . after an incubation at 37 ° c . for 30 min these cells were combined with those infected previously . the respective cell suspensions were plated on an agar plate ( 20 × 20 cm ) with lb / chloramphenicol medium and generally contained between 10 5 and 10 7 colony forming clones . the plates were incubated for 16 hours at 37 ° c ., the colonies grown were floated off with 10 ml of soc medium contianing 10 % ( v / v ) glycerol and stored in aliquots of 1 . 0 ml each at − 80 ° c . for repeated phage production and new cycles of affinity enrichment the method described was repeated selecting ten times lower culture volumes for the culture of the bacterial cells and more stringent washing conditions ( 2nd round : 3 × washing with pbst , three times of incubation with 4 % bsa in pbs for 5 min and 3 × washing with pbs ; 3rd round : 6 × washing with pbst 0 . 1 , three times incubation with 4 % of bsa for 5 min and 3 × washing with pbs ). isolation and characterization of monoclonal phagemids with specific binding to the target substrates ( single phage elisa ) from each of the clones obtained after the 3rd round of the affinity enrichment on recglp1 - r , f c igm as well as hc , 96 were selected randomly and analyzed in a single phase elisa with respect to their binding to the respective antigen or hapten , respectively . for this purpose , 300 μl each of 2 × yt / chloramphenicol medium were inoculated with a single colony and agitated for 18 hours at 37 ° c . and 220 rpm . with 80 μl each of this stationary culture 4 ml 2 × xt / chloramphenicol medium were inoculated and agitated for 4 hours at 37 ° c . and 180 rpm . to enable a parallel operation 4 “ deep well ” plates ( qiagen , each having 24 wells ) were used for this purpose . after infection of the xl1 blue cells with each 10 11 cfu m13ko7 helper phages an incubation was carried out for 30 min at 37 ° c . after additional 30 min at 37 ° c . and 180 rpm the addition of 50 mg / l kanamycin was performed . then , agitation was continued for 30 min at 37 ° c . and 220 rpm and the gene expression on pmubi - 1 was induced by adjusting the culture to 0 . 2 mg / l anhydro - tetracycline ( stock solution : 1 . 0 mg / ml in dmf ). the incubator temperature was then decreased to 22 ° c ., and the culture was agitated for 16 hours at 180 rpm . the cells were finally sedimented by centrifugation ( 30 min , 5 , 000 g , 4 ° c . ), and the supernatants were transferred into fresh “ deep well ” plates . the phagemids contained were precipitated by addition of 1 volume of 20 % ( w / v ) peg 6 , 000 , 2 . 5 m nacl and incubation for 1 hour on ice and sedimented by centrifugation ( 30 min , 5 , 000 g , 4 ° c .). the phagemids were then dissolved in 1 . 0 ml of sterile ice - cold pbs and mixed 1 : 1 with 6 % pbst and incubated for 1 hour at rt . for carrying out the elisa one well of a microtiter plate per each monoclonal phagemid to be analyzed was filled with antigen at 4 ° c .— and one was filled with bsa solution . for the saturation of the remaining binding sites on the plastic surface each of the wells was blocked by 3 % bsa ( w / v ) pbst 0 . 5 . afterwards , the wells were rinsed three times with pbst 0 . 1 and tapped out . then , 100 μl each of the phagemid solutions prepared above were pipetted into the respective wells of the plate . after an incubation period of 2 hours washing was carried out three times with pbst 0 . 1 . for the detection of bound phagemids an m13 antibody peroxidase conjugate ( amersham pharmacia biotech ) was diluted in a ratio of 1 : 5 , 000 in pbst 0 . 1 , and 100 μl were added to each of the wells . after 1 hour of incubation at rt the wells were rinsed three times with pbst 0 . 1 , then three times with pbs . finally , 100 μl each of the immunopure kit ( pierce ) were pipetted into the wells , and the color reaction was then stopped after 15 min by addition of 100 μl of 2 m h 2 so 4 . the extinction was measured by means of a sunrise remote reader ( tecan ) at 450 nm . the dna of ubiquitin variations from phagemids which in the elisa showed a relatively strong binding signal to the respective antigen — but not to bsa —( approximately 20 ) was sequenced by means of primer seq id no . 14 and the method described above . a portion of the dna sequences analyzed exhibited shifts of the reading frame or amber stop codons and were thus not further used . amino acid substitutions of ubiquitin variations which were obtained in this manner and further analyzed are exemplarily listed in table 1 . the provision of an expression construct for the in vitro transcription / translation as well as the selection of binding proteins by means of ribosomal display was done according to schaffitzel et al . ( 2001 ). however , in contrast to this no library of antibody fragments was used in the present case but libraries of ubiquitin variations analogous to those described in example 2 . one of these libraries was prepared on the basis of the dna sequence ( seq id no . 1 ) for a modified ubiquitin protein backbone having the substitutions ile44ala , lys48arg , arg54leu , val70ala , arg72leu , gly75ala , as well as the deletion of gly76 . a second library was prepared on the basis of the dna sequence ( seq id no . 15 ) for a modified ubiquitin protein backbone carrying the substitution phe45trp . in a first step , the synthetic genes for the ubiquitin variations representing the library which were mutagenized at 8 codons according to example 2 were prepared on the basis of seq id no . 1 or seq id no . 15 , respectively , by means of pcr . this was performed in a volume of 50 μl using pfu polymerase ( promega ). for this purpose , 5 μl of the provided 10 × pfu buffer as well as 4 μl dntp mix were used and filled up with h 2 o . furthermore , the sample contained 2 . 5 μl of each of the flanking primers ( seq id no . 16 and seq id no . 17 for the library on the basis of seq id no . 1 as well as seq id no . 18 and seq id no . 19 for the library of seq id no . 15 ; 10 μm ) for the introduction of the desired base pair substitutions . as a template 1 . 0 ng of each of the plasmid dnas was used which carried the non - mutated synthetic ubiquitin gene seq id no . 1 or seq id no . 15 . after addition of 2 . 5 u of pfu polymerase an incubation was carried out in 25 cycles for 1 min each at 94 ° c ., 1 min at 65 ° c . and for 1 . 5 min at 72 ° c . a final incubation was performed for 5 min at 72 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). the spacer which comprises a portion of the envelope protein iii of bacteriophage m13 and which in the course of the in vitro translation pushes the nascent protein out of the ribosomal channel to thus enable an optimal presentation of the ubiquitin variation was synthesized in two successive pcr reactions . this was first done in a total volume of 50 μl using 5 μl of the provided 10 × pfu buffer , 4 μl dntp mix and an appropriate amount of h 2 o . furthermore , the sample contained 2 . 5 ml each of the flanking primers ( seq id no . 20 and seq id 21 ; 10 μm ) as well as 1 . 0 ng dna of the bacteriophage m13 . after addition of 2 . 5 u of pfu polymerase the incubation was performed in 25 cycles each of 1 min at 94 ° c ., 1 min at 65 ° c . and for 1 . 5 min at 72 ° c . a final incubation was done for 5 min at 72 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis , the qiaquick gel extraction kit ( qiagen ) and served as a template for the second pcr reaction . this was performed using pfu polymerase ( promega ) in a volume of 50 μl . for this purpose 5 μl of the supplied 10 × pfu buffer as well as 4 μl dntp mix were used and filled up with h 2 o . furthermore , the sample contained 2 . 5 μl of each of the flanking primers ( seq id no . 22 and seq id no . 21 ; 10 μm ). as a template , 1 . 0 ng each of the dna from the previous pcr reaction was used . after addition of 2 . 5 u of pfu polymerase the incubation was carried out in 25 cycles each for 1 min at 94 ° c ., 1 min at 55 ° c . and for 1 . 5 min at 72 ° c . a final incubation was done for 5 min at 72 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). in the following step the synthetic genes for the ubiquitin variations representing the library which had been mutagenized at 8 codons were fused to the spacer dna prepared by means of a linking pcr reaction . this was performed by means of pfu polymers ( promega ) in a volume of 50 μl . for this purpose , 5 μl of the supplied 10 × pfu buffer as well as 4 μl dntp mix were used and filled up with h 2 o . furthermore , the sample contained 2 . 5 μl of each of the flanking primers ( seq id no . 23 and seq id no . 21 ; 10 μm ). as a template , 500 ng each of the library dna and 500 ng spacer dna from the previous pcr reaction were used . after the addition of 2 . 5 u of pfu polymerase the incubation was carried out in 25 cycles each for 1 min at 94 ° c ., 1 min at 55 ° c . and for 1 . 5 min at 72 ° c . a final incubation was done for 5 min at 72 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). in the final step appropriate regulative sequences ( t7 promoter , ribosomal binding site ) were introduced for the in vitro transcription / translation reaction by means of a further pcr reaction and the linear expression construct was completed . the reaction was carried out in a sample volume of 500 μl wherein approx . 500 ng of the product obtained in the previous pcr reaction was employed and taq polymerase was used . the reaction sample — adapted to the tenfold volume — was pipetted as carried out in example 2 from 10 × taq buffer , 25 mm mgcl 2 , dntp mix as well as the flanking primers ( seq id no . 24 , seq id no . 21 ; 10 μm ). after filling up with h 2 o 2 . 5 u of taq polymerase were added and the pcr program was started . in 25 cycles the incubation was carried out for each 1 min at 94 ° c ., 1 min at 65 ° c . and for 1 . 5 min at 72 ° c . a final incubation was carried out for 5 min at 72 ° c . the desired pcr product was isolated by means of preparative agarose gel electrophoresis , the qiaquick gel extraction kit ( qiagen ) and could be used directly for the in vitro transcription / translation reaction . for the in vitro transcription / translation reaction the rts100 e . coli kit ( roche cat . no . 3186 148 ) was used according to the manufacturer &# 39 ; s instructions . for this purpose , 24 . 0 μl of e . coli lysate , 20 . 0 μl of the reaction mix , 24 . 0 μl of amino acid mix ( without met ), 2 . 0 μl methionine , 10 . 0 μl reaction buffer ( 10 ×), 2 . 0 μl mgac ( 500 mm ), 2 . 0 ml anti_ssra dna ( 200 μm ) as well as 20 . 0 μl of the respective dna library ( 0 . 5 - 1 . 0 μg ) were cautiously mixed by means of the pipette and incubated for 60 min at 30 ° c . or 37 ° c . the following steps were carried out in the cool room or on ice , respectively . the sample was cooled for 5 min on ice and the reaction was stopped by the addition of 400 μl wbt ( 50 mm tris / hcl ( ph 7 . 5 ), 150 mm nacl , 50 mm mgac , 0 . 1 % tween 20 ) containing 3 % bsa . this buffer may optionally contain 2 . 5 mg / ml heparin ( sigma ). the insoluble portions of the sample were sedimented for 5 min at 13 , 000 rpm and 4 ° c . wherein the ternary complexes consisting of the ubiquitin variation , the corresponding mrna and the ribosome remained in the supernatant . as an affinity matrix for the isolation of ternary ribosomal complexes with ubiquitin variations presented on the surface which should bind to previously defined target substances 2 wells of a microtiter plate were coated with the coresponding substance . as the target substances served recombinantly prepared growth factor vegf which was immobilized over night at 4 ° c . on the microtiter plate . unoccupied binding sites on the surface of the microtiter plate were blocked by incubation of the wells each with 400 μl 3 % bsa in wbt for 90 min at rt . after removal of the blocking solution 250 μl of the solution containing the ternary ribosomal complexes from the in vitro transcription / translation reaction were added per well and incubated for 60 min at 4 ° c . and 50 rpm . unbound complexes were then removed by washing five times with e . g . wbt ( 300 μl per well , 3 min , 4 ° c ., 50 rpm ) and vigorous tapping . complexes bound to the affinity matrix were finally split by means of 100 ml eb ( 50 mm tris / hcl ( ph 7 . 5 ), 1 . 5 m nacl , 20 mm edta )— this buffer may optionally contain 50 μg / ml rna from yeast ( sigma )— per well and incubation for 5 min at 4 ° c . and 50 rpm into mrna , protein and the ribosomal subunits . the isolation of rna from the suspension obtained was carried out as rapid as possible at room temperature and by using the rnaeasy kit of qiagen ( cat . nr . 74104 ) according to the manufacturer &# 39 ; s instructions . in this case , the elution of the rna was carried out in the final step with 30 μl rnase - free h 2 o . for the degradation of dna of linear expression constructs which might have possibly been carried over the rna solution thus obtained was treated with dnase i ( invitrogen ). for this purpose , an addition of 3 μl of dnase buffer ( 10 ×) and 3 μl dnase i ( 1 u / μl ) and an incubation for 15 min at room temperature were carried out . the dnase was then inactivated by the addition of 3 μl of 25 mm edta and incubation for 10 min at 65 ° c . the rna thus obtained was thereafter complemented by means of the reverse transcriptase reaction . for this purpose , first 8 . 5 μl of the rna were mixed with 0 . 5 μl of the oligodeoxynucleotide t7te ( seq id no . 21 ; 100 μm ) and 4 . 0 μl dntp ( 2 . 5 mm each ), incubated for 5 min at 65 ° c . and then placed on ice for 1 min . then 4 . 0 μl rt buffer ( 5 ×), 1 . 0 μl dtt ( 0 . 1 m ), 1 . 0 μl rnasin ( promega ) and 1 . 0 μl ss reverse transcriptase iii ( 200 u / μl ; invitrogen ) were added as further components and incubated for 60 min at 55 ° c . the reaction sample from the reverse transcriptase reaction was used directly in a pcr for the new re - amplification of the genetic information of enriched ubiquitin variations as well as for the reintroduction of the regulative sequences for a new selection cycle . for this purpose , two successive pcr reactions were carried out using the expand high fidelity pcr system ( roche ) in a total volume of 50 μl . for this purpose , 5 μl of the supplied 10 × buffer ( including 15 mm mgcl 2 ) as well as 4 μl dntp mix were used and filled up with h 2 o . furthermore , the sample contained 2 . 5 μl each of flanking primers ( seq id no . 23 and seq id no . 21 ; 10 μm ) as well as 2 . 5 μl of dimethylsulfoxide ( dmso ). as the template either 2 . 5 or 5 . 0 μl of the previous rt reaction was used . after the addition of 0 . 75 μl polymerase mix ( 3 . 5 u / μl ) an incubation was carried out in 25 cycles each for 15 sec at 94 ° c ., 30 sec at 50 ° c . and for 1 . 0 min at 72 ° c . a final incubation was done for 7 min at 72 ° c . the desired pcr product was isolated by means of a preparative agarose gel electrophoresis as well as the qiaquick gel extraction kit ( qiagen ) and could directly be employed for the second pcr reaction . therefore , 5 μl of the supplied 10 × buffer ( including 15 mm mgcl 2 ) as well as 4 μl dntp mix were used and filled up with h 2 o . furthermore , the batch contained 2 . 5 μl each of flanking primers ( seq id no . 24 and seq id no . 21 ; 10 μm ). as the template the total dna from the previous pcr reaction was used . after addition of 0 . 75 μl polymerase mix ( 3 . 5 u / μl ) an incubation was carried out in 25 cycles each for 15 sec at 94 ° c ., 30 sec at 50 ° c . and for 1 . 0 min at 72 ° c . a final incubation was done for 7 min at 72 ° c . a final incubation was done for 7 min at 72 ° c . the desired pcr product was isolated by means of a preparative agarose gel electrophoresis as well as the qiaquick gel extraction kit ( qiagen ) and could directly be employed for the in vitro transcription / translation of a new selection cycle . alternatively , the cloning of the respective gene into expression vector pet20b (−) ( see above ) was carried out . this enables the analysis of ubiquitin variations obtained with respect to their dna sequence , the recombinant preparation thereof in e . coli as well as the one - step purification thereof by means of immobilized metal chelate affinity chromatography ( imac ) using a carboxyterminally fused hexa - histidine peptide ( see below ). amino acid substitutions of ubiquitin variations which were obtained in this manner and further analyzed are exemplarily listed in table 2 . the characterization of the proteinchemical properties of selected ubiquitin variations from phagemids giving a relatively strong binding signal in the elisa and with functional dna sequence was carried out after cloning of the respective gene into expression vector pet20b (−) ( see above ). this enabled the recombinant preparation of the respective variation by means of e . coli bl 21 / pubs as well as the one - step purification thereof by means of immobilized metal chelate affinity chromatography ( imac ) using a carboxyterminal hexa - histidine peptide . for the recombinant preparation of the ubiquitin - based modified proteins having novel binding characteristics 50 ml of 2 × yt / amp / kan medium were inoculated with a respective single colony and agitated for 16 hours at 37 ° c . and 220 rpm . with this pre - culture 1 . 5 liters of 2 × yt / amp / kan medium was inoculated in a ratio of 1 : 50 and incubated at 37 ° c . and 220 rpm until a cell density of od 600 = 0 . 5 was reached . after induction of the expression of the foreign gene by addition of 1 mm / 1 α - d - isopropyl thiogalactoside ( iptg ) agitation was continued for further 3 hours at 37 ° c . and 220 rpm . the cells were then sedimented by centrifugation ( 30 min , 5 , 000 g , 4 ° c .) and resuspended in 40 ml npi - 20 ( 50 mm nah 2 po 4 , ph 8 . 0 , 300 mm nacl , 20 mm imidazole ). the cell disruption was done by incubation for half an hour with 200 μg / ml lysozyme and 500 u of benzonase at rt as well as a five times of ultrasonic pulsing each for 15 sec . the cell debris was sedimented by centrifugation ( 30 min , 15 , 000 g , 4 ° c .) and the supernatant containing the total soluble cell protein could directly be employed in the subsequent imac . the chromatography was performed on an äkta ™ explorer fplc unit ( amersham pharmacia biotech ) using a 5 ml hitrap chelating hp column ( amersham pharmacia biotech ) at rt and a flow rate of 5 ml / min . first , the column was equilibrated with 5 column volumes of npi - 20 whereupon the total cell protein was passed over the column . unbound protein was washed off by rinsing with 30 column volumes of npi - 20 . the elution was done with a linear gradient of 20 nm to 250 mm of imidazole in a total of 20 column volumes while the eluate was collected in fractions of 2 . 5 ml each . the fractions containing the purified ubiquitin variation were analyzed by means of sds polyacrylamide gel electrophoresis and combined . the yields of the recombinant ubiquitin variations achieved were between 10 and 30 mg / l of culture volume . the binding of the ubiquitin variations selected in each case and which were purified as described in example 6 on recglp1 - r , f c igm , and hc was detected in the elisa with either an ni / nta peroxidase conjugate ( qiagen ) or with an ubiquitin antiserum ( sigma ) from rabbit . for this purpose , the wells of microtiter plates were filled over night at 4 ° c . with the respective antigen and e . g . bsa for the detection of unspecific binding and blocked for 2 hours with 3 % bsa ( w / v ) pbst 0 . 5 for the saturation of the remaining binding sites . the plates were then rinsed three times with pbst 0 . 1 and tapped out . thereafter , 100 μl of each of the solutions of the respective modified purified protein in pbst 0 . 1 in serial concentrations ( ranging from undiluted up to a dilution of 1 : 16 ) were pipetted into the wells of the plate . the incubation period of 2 hours was followed by rinsing three times with pbst 0 . 1 . for the detection of the bound modified protein the ni / nta peroxidase conjugate was diluted in a ratio of 1 : 500 , or the ubiquitin antiserum was diluted in a ratio of 1 : 10 in pbst 0 . 1 , respectively , and 100 μl each were added to each of the wells . the incubation for one hour at rt was either directly followed by the detection of the ni / nta peroxidase conjugate ( see below ) or by the incubation with an rabbit antibody peroxidase conjugate ( 1 : 2 , 500 in pbst 0 . 1 ), respectively , for one hour at rt . for detection the wells were rinsed three times with pbst 0 . 1 , then three times with pbs . finally , 100 μl each of the immunopure kit were pipetted thereto and the color reaction was then stopped after 15 min by addition of 100 ml of 2 m h 2 so 4 . the extinction measurement the was done by means of a sunrise remote reader ( tecan ) at 450 nm . the values of the absorption intensity obtained were evaluated by means of the “ sigma plot ” computer program . for this purpose , the extinction measured in each case was plotted against the corresponding protein concentration employed , and the curve obtained was fitted by means of non - linear regression using formula ( 1 ). y = a * x ( b + x ) ( 1 ) assuming an association / dissociation equilibrium between the immobilized antigen and the modified protein thus is : y = concentration of the complex of antigen / modified protein ( here measured indirectly via the enzymatic activity of the reporter enzyme ) the binding curves obtained from an elisa experiment of this type for the ubiquitin variation spu - 1 - d10 which had been obtained from the affinity enrichment on recglp1 - r by means of phage display according to examples 3 and 4 are exemplarily illustrated in fig5 . furthermore , in fig6 is illustrated the binding curves of the ubiquitin variation spw - 11 - a1 which resulted from the affinity enrichment to vegf by means of ribosomal display according to example 5 . the binding data obtained for the individually selected ubiquitin - based modified proteins having novel binding properties are summarized in table 3 . for a quantitative analysis of the binding of variations of ubiquitin to a previously defined antigen a biacore 3000 system ( biacore ) was also used . for such surface plasmon resonance ( spr ) measurements a carrier ( sensor chip ) capable of immobilizing the analyte was positioned within the device and coupled to the microflow system ( integrated μfluidic cartridge , ifc ) integrated therein . this enabled a continuous fluid flow from the buffer reservoir across the chip surface and a quasi in solution detection of the binding to the analyte carrier . first , for a measurement the target molecule , e . g . vegf , was coupled by means of nhs / edc coupling via primary amine groups to the carboxymethyl dextrane surface of the sensor chip cm5 ( biacore ) according to the manufacturer &# 39 ; s instructions . the measurements were carried out in a continuous buffer flow of 35 μl / min at 25 ° c . as a running buffer and a solvent for the proteins used pbs ( filter sterilized and degassed ) supplemented with 0 . 005 % p - 20 detergent ( biacore ) was used . the interaction with the immobilized vegf of different concentrations of the ubiquitin variations which were injected via the sample loop could be detected due to the generated spr signal . the signal was quantified as so - called resonance units ( ru ) depending on the time and corresponding binding curves were generated . the binding curves obtained were evaluated using the biaevaluation software ( version 3 . 1 ; biacore ), and the value of the dissociation constant ( k d ) was determined . the binding curves of the ubiquitin variation spu - 11 - 58 which resulted from the affinity enrichment to vegf by means of ribosomal display according to example 5 obtained from such a biacore experiment are exemplarily illustrated in fig7 . the binding data obtained for the individual selected ubiquitin - based modified proteins having novel binding properties are summarized in table 3 . furthermore , individual modified proteins were evaluated with respect to the specificity of their binding . for this purpose , elisa experiments were carried out as described above using a single appropriate concentration of the modified proteins . for this purpose , respective wells of the microtiter plate were filled with the target substrate as well as structurally similar substances . the binding curves of ubiquitin variation spu - 3 - h13 which resulted from the affinity enrichment to hc according to examples 3 and 4 obtained in such an elisa experiment are exemplarily illustrated in fig8 . improvement of the binding characteristics of ubiquitin - based binding proteins by site - directed secondary mutagenesis based on the ubiquitin variation spu - 2 - a7 which shows a de novo binding property against f c igm a generally applicable strategy for the affinity maturation was established . this comprised first a new random substitution of selected positions in the binding site generated de novo on the dna level as well as the subsequent parallel expression , purification and binding analysis of 96 of the respective variations . by the use of this “ 96 format ” it is possible to transfer the strategy to laboratory roboters and thus to carry out the analysis of variations with high throughput . for this purpose , the codons of each of two positions ( 62 and 63 as well as 64 and 65 ) were randomly mutated by means of pcr in two parallel samples . the corresponding reactions were carried out each in 50 μl wherein approximately 1 . 0 ng of the gene for spu - 2 - a7 inserted into pet20b (−) were used and taq polymerase was employed . the reaction sample was pipetted as described in example 2 from 10 × taq buffer , 25 mm mgcl 2 , dntp mix as well as the flanking primers ( seq id no . 12 , seq id no . 25 for the mutation of positions 62 and 63 or seq id no . 12 , seq id no . 26 , respectively , for the mutation of positions 64 and 65 ; 10 μm ). after filling up with h 2 o 2 . 5 u of taq polymerase were added and the pcr program was started . in 25 cycles the incubation was carried out each for 1 min at 94 ° c ., 1 min at 55 ° c . and for 1 . 5 min at 72 ° c . a final incubation was carried out for 5 min at 72 ° c . the desired pcr products were isolated by means of preparative agarose gel electrophoresis and the qiaquick gel extraction kit ( qiagen ). the dna - fragments obtained each representing the libraries of spu - 2 - a7 mutagenized at positions 62 / 63 or 64 / 65 , respectively , were cut by means of preparative ndei / xhoi restriction cleavage and isolated again by means of preparative agarose gel electrophoresis . the insertion of the genetic libraries for the modified spu - 2 - a7 was carried out into expression vector pet20b (−) ( novagen , c . f . example 1 ) for the production of the corresponding protein . following the transformation of electrocompetent e . coli , e . g . nova - blue or bl - 21 cells , single colonies were obtained which contained the genetic information of one clone each of the library obtained in the form of the expression plasmid . using 96 of these single colonies 300 μl 2 × yt / amp / kan each were inoculated and agitated over night at 37 ° c . and 220 rpm . with 100 μl of each of said cultures 96 × 4 ml of 2 × yt / amp / kan were inoculated and incubated at 37 ° c . and 220 rpm until a cell density of od 600 = 0 . 5 was reached . for this purpose , four 24 - well culture plates ( qiagen ) were used in each case . after induction of the expression of the foreign gene by addition of 1 mm / l α - d - isopropyl thiogalactoside ( iptg ) agitation was continued for additional 3 hours at 37 ° c . and 180 rpm or over night at 30 ° c . and 180 rpm . the cells were then sedimented by centrifugation ( 30 min , 4 , 000 g , 4 ° c .) and the supernatant was removed . the cell disruption was carried out by physical ( shock freezing and thawing ) or chemical lysis ( detergents ) as well as by addition of lysozyme ( 200 μg / ml ) and benzonase ( 10 u / ml in the final volume ). the purification of the variations of spu - 2 - a7 from the total cell protein obtained was carried out by means of the biorobot 9600 kit from qiagen and a manually operated vacuum station ( qiavac 96 , qiagen ). the protein solutions obtained were analyzed with respect to their binding to f c igm in a qualitative manner in an elisa experiment corresponding in its experimental set - up to that of example 7 . for the verification of the functionality of their gene or the analysis of the substitutions obtained , respectively , variations of spu - 2 - a7 showing a relatively strong binding signal in the elisa were subjected to dna sequencing . promising candidates were prepared on an 1 . 5 l scale in a recombinant manner and purified by means of immobilized metal chelate affinity chromatography ( imac ) using the hexa - histidine peptide fused to the carboxyterminus . for the quantification of the affinity a concentration - independent elisa as described in example 7 was carried out in comparison to spu - 2 - a7 — the ubiquitin variation on the basis of which the maturation had been performed . the binding curves of the ubiquitin variation spu - 2 - a7 as well as of spu - 2 - a7 ( 62 / 63 ) which were obtained from such an elisa experiment are exemplarily illustrated in fig9 . with a k d = 1 . 0 μm this affinity - maturated variation exhibits a binding strength improved by a factor 10 in comparison to sp - 2 - a7 . site - directed coupling of two identical ubiquitin - based proteins via single carboxy - terminal cysteine residues by means of bis - maleimide reagents the linkage of two identical ubiquitin variations was carried out via cysteine residues which were specifically introduced for this purpose by means of bis - maleimido hexane ( bmh , pierce ). first , an appropriate codon for cysteine followed by a proline was introduced for this purpose at the 3 ′ end of the reading frame — i . e . at the protein carboxyterminus after the hexa - histidine peptide . in this way , the last proline residue should protect the resulting protein from proteolytic degradation during production and purification . the insertion into pet20b (−) of the corresponding base pairs was carried out by means of the quick change ™ site - directed mutagenesis kit ( stratagene ) according to the manufacturer &# 39 ; s instructions and by using the oligodeoxynucleotides seq id no . 27 and seq id no . 28 as well as the gene of an ubiquitin variation ( seq id no . 15 ) as a template . the resulting clones were analyzed by means of dna sequencing and colonies having the expression plasmid carrying the desired insertion were used for recombinant production and purification according to example 6 . the purified protein was dialyzed against pbs containing 1 mm edta and 5 mm β - mercaptoethanol and employed in a concentration of approx . 1 mg / ml for the following coupling reaction in which pbs , 1 mm edta served as the reaction buffer . for this purpose , the free cysteine residue was first reduced by adjusting the protein solution to 100 mm dithiothreitol ( dtt ) and incubation for 1 hour at room temperature . thereafter , the reducing reagents were separated from the protein by means of a pd10 sephadex g - 25 m column ( amersham biosciences ), this was added with a twofold molar excess of coupling reagent ( stock solution : 100 mm bmh in dmso ) and incubated for one hour at room temperature . excess bmh was separated again using a pd10 column . to separate only singly reacted coupling reagent which might possibly be present an equimolar amount of freshly reduced protein was again added to the coupling sample thus obtained . this second coupling step was carried out for 2 hours at room temperature . the coupling sample obtained by this procedure was analyzed by means of sds - 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