Patent Application: US-18101205-A

Abstract:
the invention relates to the field of aqueous solutions comprising a protein . more specifically , the invention relates to the detection and / or removal of conformationally altered proteins and / or peptides comprising a cross - β structure from an aqueous solution comprising a protein . the invention provides methods for detecting and / or removing proteins and / or peptides comprising a cross - β structure from an aqueous solution comprising a protein , the method comprising contacting the aqueous solution comprising a protein with at least one cross - β structure - binding compound resulting in a bound protein or peptide with cross - β structure . the invention further provides a aqueous solution comprising a protein obtainable by a method of the invention , and a kit for carrying out the methods of the invention .

Description:
examples of useful applications of a method according to the invention are provided above and even more examples are provided below . in general it can be said that if one wants to study or obtain a protein with a particular property , it is important to check each and every treatment on their cross - β structure inducing capabilities on the protein . if , for example , a protein is used in the food industry or as a biochemical compound in research ( for example , biomedical research , or in diagnostics it is important to check the production , purification and storage conditions . if one wants to study the activity of a protein ( for example , an enzyme ) it is important to study all the conditions to which such a protein is subjected . other , non - limiting , applications of a method according to the invention are : testing of conditions for producing , purifying and storing proteins used for growing crystals for protein crystallography purposes ; some of the presently used conditions result in the formation of cross - β structure in a protein and hence hamper the growth of high - quality crystals of the protein ; conditions ( to be ) used in crystallography are now tested for their cross - β structure inducing capability and a selection is made for conditions that do not or only slightly induce the formation of cross - β structure in a protein ; testing of chemical / biochemical / biophysical conditions used in protein purifications ; independent of the source of protein ( naturally expressed or recombinantly expressed ) proteins are typically subjected to one or multiple purification steps to obtain high grade preparations comprising a protein and / or peptide . all treatments performed with a protein or peptide in such purifications , such as buffer composition , temperature , column material , dialysis membranes , membranes used for concentration , is checked with a method according to the invention and conditions are selected that do not or only slightly induce cross - β structure formation in the to be purified protein ; testing of conditions and / or solutions for protein refolding from an aggregated state to a native fold ; independent of the source of the protein with non - native fold ( naturally expressed or recombinantly expressed ; for example , escherichia coli inclusion bodies ), proteins are typically subjected to exposure to one or more solutions that putatively aid the folding from a non - native fold to a native fold . the solutions are now checked with a method according to the invention for their propensity to induce the cross - β structure in proteins by testing the content of cross - β structure in the proteins after the exposure to the solutions . solutions can now be selected that do not result in cross - β structure and thus may aid the adoption of a native fold ; selection and development of cell culture solutions or laboratory liquid equipment comprising proteins or peptides in general . it is revealed in the specification that several physical / chemical conditions influence the fold of a protein . exposure to cl or dxs500k , a freeze - thaw cycle , variations in protein purification protocol , heating , change in ph , the source of the protein and exposure to plastic all introduce a structural rearrangement in the protein accompanied by the formation of the amyloid - like cross - β structure fold . this new fold can be detected by , amongst others , tpa binding , tpa activation , factor xii binding and by conventional amyloid fluorescence assays . the invention is further explained in the examples , without being limited by them . a ) antigen albumin - age and ligand aβ were send in to davids biotechnologie ( regensburg , germany ); a rabbit was immunized with albumin - age , antibodies against a structural epitope were affinity purified using a column with immobilized aβ . b ) ppack is phe - pro - arg - chloromethylketone , εaca is ε - amino caproic acid , tpa is tissue - type plasminogen activator for preparation of advanced glycation end - product ( age )- modified bovine serum albumin , 100 mg ml − 1 of albumin was incubated with phosphate buffered saline ph 7 . 3 ( pbs ) containing 1 m of glucose - 6 - phosphate ( g6p ) and 0 . 05 % m / v nan 3 , at 37 ° c . in the dark . glycation was prolonged up to 23 weeks . 5 to prepare glycated hemoglobin ( hb - age ), human hemoglobin ( hb , sigma - aldrich , h7379 ) at 5 mg ml − 1 was incubated for 32 weeks at 37 ° c . with pbs containing 1 m of g6p and 0 . 05 % m / v of nan 3 . in control solutions , g6p was omitted . after incubations , solutions were extensively dialyzed against distilled h 2 o and , subsequently , stored at 4 ° c . protein concentrations were determined with advanced protein - assay reagent adv01 ( cytoskeleton , denver , colo ., us ). glycation and formation of age was confirmed by measuring intrinsic fluorescent signals from age ; excitation wavelength 380 nm , emission wavelength 435 nm . in addition , binding of age - specific antibodies was determined . presence of cross - β structure or cross - β structure conformation in albumin - age was confirmed by enhancement of congo red fluorescence , enhancement of thioflavin t ( tht ) fluorescence , the presence of β - sheet secondary structure , as observed with circular dichroism spectropolarimetry ( cd ) analyzes , and by x - ray fiber diffraction experiments . 5 presence of cross - β structures in hb - age was confirmed by tpa binding , cd analyses , transmission electron microscopy ( tem ) imaging of fibrillar structures and by congo red fluorescence measurements . amyloid preparations of human γ - globulins were made as follows . lyophilized γ - globulins ( g4386 , sigma - aldrich ) were dissolved in a 1 (:) 1 volume ratio of 1 , 1 , 1 , 3 , 3 , 3 - hexafluoro - 2 - propanol and trifluoroacetic acid and subsequently dried under an air stream . dried 7 - globulins were dissolved in h 2 o to a final concentration of 1 mg ml − 1 and kept at room temperature for at least three days , or kept at 37 ° c . for three days and subsequently at − 20 ° c . aliquots were stored at − 20 ° c . and analyzed for the presence of cross - β structures . fluorescence of congo red and tht was assessed . in addition tpa binding was analyzed in an elisa and tpa activating properties in a chromogenic plasminogen ( plg ) activation assay . in addition , the macroscopic appearance of denatured γ - globulins was analyzed with tem imaging . human amyloid - β ( aβ ) ( 1 - 40 ) dutch type ( daefrhdsgyevhhqklvffaqdvgsnkgaiiglmvggvv ) ( seq id no : _ ), was disaggregated in a 1 : 1 ( v / v ) mixture of 1 , 1 , 1 , 3 , 3 , 3 - hexafluoro - 2 - isopropyl alcohol and trifluoroacetic acid , air - dried and dissolved in h 2 o ( 10 mg ml − 1 ). after three days at 37 ° c ., the peptide was kept at room temperature for two weeks , before storage at 4 ° c . aβ solutions were tested for the presence of amyloid conformation by tht or congo red fluorescence and by tem imaging . negative control for cross - β structure detection assays was non - amyloid fragment fp10 of human fibrin α - chain ( 148 - 157 ) ( krlevdidik ) ( seq id no : _ ). 16 , 17 fp10 was dissolved at a concentration of 1 mg ml − 1 in h 2 o and stored at 4 ° c . this solution was used as a negative control for tht fluorescence assays . f4 - 5 domains and the f domain of tpa with a carboxy - terminal his 6 - tag were also expressed in saccharomyces cerevisiae . the cdna constructs were prepared following standard procedures , 18 by the biotechnology application center ( bac - vlaardingen / naarden , the netherlands ). domain boundaries of fn f4 - 5 and tpa f were taken from the human fn and human tpa entries in the swiss - prot database ( p02751 for fn , p00750 for tpa ) and comprised amino - acids nh 2 - 1182 - v276 - cooh of fn f4 - 5 and nh 2 - g33 - s85 - cooh of tpa . affinity purification of the expressed proteins was performed using his 6 - tag - ni 2 + interaction and a desalting step . constructs were stored at − 20 ° c . in pbs ph 7 . 0 . the molecular size of the constructs was checked on a coomassie brilliant blue - stained sds - page gel . totally chemical synthesis of the f domains of hepatocyte growth factor activator ( hgfa , swissprot entry q04756 ) and tpa ( swissprot entry p00750 ) was performed in the laboratory of dr . t . m . hackeng ( academic hospital maastricht , the netherlands ), according to standard procedures . 19 both domains were synthesized as two separate peptides that were subsequently ligated using native chemical ligation . the tpa f domain was completed with a carboxy - terminal acetylated lysine residue or biotinylated lysine residue . the hgfa f domain was supplied with an acetylated lysine residue . products were analyzed on a reversed phase hplc column and with mass spectrometry . cloning , expression and purification of the soluble extracellular domains of receptor for advanced glycation end - products the soluble extracellular part , of the receptor for age ( srage ) was cloned , expressed and purified as follows ( q .- h . zeng , prof . p . gros , dept . of crystal - & amp ; structural chemistry , bijvoet center for biomolecular research , utrecht university , utrecht , the netherlands ). human cdna of rage was purchased from rzpd ( clone iralp962e1737q2 , rzpd , berlin , germany ). for pcrs , the gagatctgctcaaaacatcacagcccgg ( seq id no : _ ) forward primer was used comprising a bglii site , and the gcggccgcctcgcctggttcgatgatgc ( seq id no : _ ) reverse primer with a noti site . the soluble extracellular part of rage comprises three domains spanning amino - acid residues 23 - 325 . the pcr product was cloned into a ptt3 vector , containing an amino - terminal his - tag and a thrombin cleavage site . the srage was expressed in 293e hamster embryonic kidney cells at the abc - protein expression facility ( utrecht university , utrecht , the netherlands ). concentrated cell culture medium was applied to a hi - trap chelating hp ni 2 + - nta column ( amersham biosciences europe , roosendaal , the netherlands ). the running buffer was 25 mm tris - hcl , 500 mm nacl , ph 8 . 0 . the protein was eluted by using a step gradient of 0 to 500 mm imidazole . purity of the his - srage was depicted from coomassie stained sds - page gels . after concentration , the buffer was exchanged to 20 mm tris - hcl , 200 mm nacl , 100 μm phenylmethylsulfonyl fluoride ( pmsf ), ph 8 . 0 . various stocks at 1 , 5 and 20 mg ml − 1 were first kept at 4 ° c . for several weeks and then stored at − 20 ° c . in this way , the pmsf will be sufficiently inactivated at 4 ° c . plasmin ( pls ) activity was assayed as described . 16 peptides and proteins that were tested for their stimulatory ability were regularly used at 100 μg ml − 1 . the tpa and plasminogen ( plg ) concentrations were 200 pm and 1 . 1 μm , respectively , unless stated differently . chromogenic substrate s - 2251 ( chromogenix , instrumentation laboratory spa , milano , italy ) was used to measure pls activity . conversion of zymogen factor xii (# 233490 , calbiochem , emd biosciences , inc ., san diego , calif .) to proteolytically active factor xii ( factor xiia ) was assayed by measurement of the conversion of chromogenic substrate chromozym - pk ( roche diagnostics , almere , the netherlands ) by kallikrein . chromozym - pk was used at a concentration of 0 . 3 mm . factor xii , human plasma prekallikrein (# 529583 , calbiochem ) and human plasma cofactor high - molecular weight kininogen (# 422686 , calbiochem ) were used at concentrations of 1 μg ml − 1 . the assay buffer contained hbs ( 10 mm hepes , 4 mm kcl , 137 mm nacl , 5 μm zncl 2 , 0 . 1 % m / v albumin ( a7906 , sigma , st . louis , mo ., usa ), ph 7 . 2 ). assays were performed using microtiter plates ( costar , cambridge , mass ., usa ). peptides and proteins were tested for their ability to activate factor xii . 150 μg ml − 1 kaolin , an established activator of factor xii was used as positive control and solvent ( h 2 o ) as negative control . the conversion of chromozym - pk was recorded kinetically at 37 ° c . for at least 60 minutes . assays were done in duplicate . in control wells factor xii was omitted from the assay solutions and no conversion of chromozym - pk was detected . in some assays albumin was omitted from the reaction mixture . alternatively , chromogenic substrate s - 2222 ( chromogenix ) was used to follow the activity of factor xii itself . with s - 2222 , activation of factor xii in plasma was measured , using 60 % v / v plasma , diluted with substrate and h 2 o with or without potential cofactor . furthermore , auto - activation of factor xii was measured by incubating 53 μg ml − 1 purified factor xii in 50 mm tris - hcl buffer ph 7 . 5 with 1 mm edta and 0 . 001 % v / v triton - x100 , with s - 2222 and h 2 o with or without potential cofactor . binding of cross - β structure containing peptides / proteins was studied using surface plasmon resonance technology with a biacore 2000 apparatus ( biacore ab , uppsala , sweden ). a standardized amine coupling procedure was used to couple proteins with f domains to a cm5 chip ( biacore ab , uppsala , sweden ). first , the dextran surface of the chips was activated by a 35 μl injection with a 1 : 1 mixture of 0 . 1 m n - hydroxysuccinimide ( nhs ) and 0 . 4 m n - ethyl - n ′-( dimethylaminopropyl ) carbodiimide ( edc ) at a flow rate of 5 μl minute − 1 . then , the proteins were covalently coupled to the activated dextran surface . remaining activated groups in each of the four flow channels were blocked by injection of 35 μl of 1 m ethanolamine hydrochloride ph 8 . 5 . edc , nhs and ethanolamine hydrochloride were obtained from biacore . on one chip , on channels 1 to 4 , buffer ( reference channel ), the soluble extracellular part of receptor for advanced glycation end - products ( srage ), tpa and k2p - tpa were immobilized . the immobilization buffer for the reference channel , channel 2 ( srage ), channel 3 ( tpa ) and channel 4 ( k2p - tpa ) was 10 mm acetate ph 3 . 75 . in channel 2 , 2000 response units ( ru ) srage was immobilized , 2700 ru and 2400 ru tpa and k2p - tpa are immobilized , respectively . the flow rate was 10 μl minute − 1 , the injection time was 120 seconds . the running buffer during immobilization was 10 mm hepes ph 7 . 4 , 140 mm nacl . buffers were filtrated on a 0 . 22 μm filter ( white gswp , 47 mm , millipore ) and degassed at room temperature . for subsequent binding experiments , the running buffer was 10 mm hepes ph 7 . 4 , 140 mm nacl , 1 . 5 mm cacl 2 , 10 mm faca , 0 . 005 % tween - 20 . binding of albumin - age was determined with a solution of 3 . 9 μg ml − 1 albumin - age in running buffer . albumin - age was filtered on a millex - gv 0 . 22 μm filter unit ( millipore ). binding of filtered hb - age was tested at 32 μg ml − 1 . binding of amyloid γ - globulins were tested at 62 . 5 μg ml − 1 . after each injection of protein , the chip was regenerated with 0 . 1 m h 3 po 4 ph 1 . 0 . after injections with albumin - age and hb - age this regeneration step was successful and sufficient , after injection with amyloid γ - globulins , the bound protein could not be released , not even after injection with more harsh regeneration buffers ( hcl , naoh ). binding of hb - age was also tested after centrifugation for 10 minutes at 16 , 000 * g alternative to filtration . tpa activation before and after filtration was assessed with a plg - activation assay . also amyloid γ - globulins and amyloid endostatin ( entremed , inc ., rockville , md ., usa ) were tested before and after centrifugation . on a second chip , buffer , chemically synthesized hgfa f domain , chemically synthesized tpa f domain and fn f4 - 5 - his6 , expressed in s . cerevisiae , were immobilized . hgfa f was immobilized in 10 mm acetate buffer ph 4 . 0 , 190 ru . tpa f was immobilized in 5 mm maleate ph 5 . 5 , 395 ru , fn f4 - 5 in 5 mm maleate ph 6 . 0 , 1080 ru . now , the running buffer was 10 mm hepes ph 7 . 4 , 140 mm nacl , 1 . 5 mm cacl 2 , 10 mm faca , 0 . 05 % tween - 20 . regeneration buffer was running buffer supplemented with 1 m nacl . binding was tested with endostatin at 0 - 800 nm , hb - age at 0 - 25 nm , recombinant β2 - glycoprotein i ( β2gpi ) at 0 - 300 nm and 25 nm native hb . for the fn f4 - 5 channel , the maximum binding expressed in ru was plotted against the concentrations . for both chips , channel 1 was used for reference purposes . the signal obtained with this channel was subtracted from the signals obtained with the channels with immobilized proteins . fluorescence of tht — protein / peptide adducts was measured as follows . solutions of 25 μg ml − 1 of protein or peptide preparations were prepared in 50 mm glycine buffer ph 9 . 0 with 25 μm tht . fluorescence was measured at 485 nm upon excitation at 435 nm . background signals from buffer , buffer with tht and protein / peptide solution without tht were subtracted from corresponding measurements with protein solution incubated with tht . regularly , fluorescence of aβ was used as a positive control , and fluorescence of fp10 , a non - amyloid fibrin fragment , 16 was used as a negative control . fluorescence was measured in triplicate on a hitachi f - 4500 fluorescence spectrophotometer ( ltd ., tokyo , japan ). solutions of 25 μg ml − 1 protein / peptide were incubated with 25 μm congo red in pbs and fluorescence was measured at 590 nm upon excitation at 550 nm . background signals from buffer , buffer with congo red and protein / peptide solution without congo red were subtracted from corresponding measurements with protein solution incubated with congo red . fluorescence was measured in triplicate on a hitachi f - 4500 fluorescence spectrophotometer ( ltd ., tokyo , japan ). for tem analysis of protein en peptide solutions grids were prepared according to standard procedures . samples were applied to 100 - mesh copper grids with carbon coated formvar ( merck , germany ), and subsequently washed with pbs and h 2 o . grids were applied to droplets of 2 % ( m / v ) methylcellulose with 0 . 4 % ( m / v ) uranylacetate ph 4 . after a 2 - minute incubation , grids were dried on a filter . micrographs were recorded at 80 kv , at suitable magnifications on a jem - 1200ex electron microscope ( jeol , japan ). formulated protein therapeutics were obtained from the local hospital pharmacy and were used as supplied by the manufacturers . the following protein therapeutics were purchased : 1 ) human growth hormone ( gh ) ( genotropin , batch 52344b51 , 5 mg ml − 1 kabiquick , pharmacia b . v ., woerden , the netherlands ), 2 ) recombinant human zn 2 + - chelated insulin ( monotard , batch ns61694 , 100 ie ml − 1 , novo nordisk , bagsvaerd , denmark ), 3 ) human albumin ( cealb , batch ns61694 , 200 mg ml − 1 , sanquin - clb , amsterdam , the netherlands ), 4 ) human - modified gelatin ( gelofusine , batch 030606h4 , 40 mg ml − 1 , braun medical bv , oss , the netherlands ), 5 ) rapid acting human insulin analogue ( novorapid flexpen , batch ph70008 , 10 u ml − 1 , novo nordisk ), 6 ) blood cell growth factor filgrastim ( neupogen singleject , batch n0693ad , 960 μg ml − 1 , amgen europe , breda , the netherlands ), 7 ) human - murine chimeric monoclonal antibody ( remicade - infliximab , batch 03d06h120a , 10 mg ml − 1 , centocor , leiden , the netherlands ), 8 ) abciximab , an inhibitor of blood platelet aggregation ( reopro , 2 mg ml − 1 , centocor , leiden , the netherlands ) and 9 ) human coagulation factor viii ( fviii ) isolated from healthy volunteers ( aafact , lot 02l046250a , 3 . 6 mg ml − 1 , sanquin - clb , amsterdam , the netherlands ). lyophilized therapeutics were dissolved according to the manufacturers recommendations . gh , zinc - insulin , cealb and gelatin were stored at − 20 , 4 , room temperature , 37 and 65 ° c . other protein therapeutics were only kept at 4 ° c ., and assayed for the presence of cross - β structure at shown time points . enhancement in fluorescence of tht and congo red was measured with all formulated protein therapeutics . for this purpose , proteins were diluted to the indicated concentrations . in addition , tpa binding to the protein therapeutics was analyzed by elisa and activation of tpa was tested using the plg - activation assay . zinc - insulin was diluted tenfold in the activation assay , gh was diluted to a final concentration of 500 μg ml − 1 . activation of factor xii and prekallikrein by the therapeutics was tested in the chromogenic factor xii assay ( see above ). for tpa elisas , 5 μg ml − 1 of the protein therapeutics were coated onto greiner high - binding microlon plates (# 655092 , greiner bio - one , alphen a / d rijn , the netherlands ). after coating , plates were blocked with blocking reagent ( roche diagnostics , almere , the netherlands ). a concentration series of tpa or k2p - tpa in pbs with 0 . 1 % v / v tween - 20 and 10 mm e - amino caproic acid was applied and the plates were incubated for one hour at room temperature with constant swirling . binding of tpa was assessed with monoclonal antibody 374b that binds to the protease domain of both tpa and k2p - tpa ( american diagnostica , tebu - bio , the netherlands ), peroxidase - conjugated rabbit anti - mouse immunoglobulins ( rampo , p0260 , dakocytomation , glostrup , denmark ), and stained with 3 ′ 3 ′ 5 ′ 5 ′- tetramethylbezidine ( tmb , catalogue number 4501103 , buffer , catalogue number 4501401 , biosource int ., camarillo , calif ., usa ). activation of tpa by β 2 - glycoprotein i , binding of factor xii and tpa to β 2 - glycoprotein i , and tht and tem analysis of p 2 - glycoprotein i purification of β 2 - glycoprotein i ( β2gpi ) was performed according to established methods . 20 , 21 recombinant human β 2 gpi was made using insect cells and purified as described . 20 plasma derived β 2 gpi as used in a factor xii elisa , the chromogenic plg - activation assay , was purified from fresh human plasma as described . 21 alternatively , β 2 gpi was purified from , either fresh human plasma , or frozen plasma (− 20 ° c .) on an anti - β 2 gpi antibody affinity column . 22 activation of tpa ( actilyse , boehringer - ingelheim ) by β 2 gpi preparations was tested in the plg - activation assay ( see above ). hundred μg ml − 1 plasma β 2 gpi or recombinant β 2 gpi were tested for their stimulatory cofactor activity in the tpa - mediated conversion of plg to pls , and were compared to the stimulatory activity of peptide fp13 . 16 binding of purified human factor xii from plasma ( calbiochem ) or of purified recombinant human tpa to β 2 gpi purified from human plasma , or to recombinant human β 2 gpi was tested in an elisa . ten μg of factor xii or tpa in pbs was coated onto wells of a costar 2595 elisa plate ( cambridge , mass ., usa ) and incubated with concentration series of the two β 2 gpi preparations . binding of β 2 gpi was assessed with monoclonal antibody 2b2 . 22 binding of factor xii to β 2 gpi was also tested using immunoblotting . β 2 gpi ( 33 μg ) purified either from fresh plasma or from frozen plasma was brought onto a 7 . 5 % sds - page gel . after blotting to a nitrocellulose membrane , the blot was incubated with 1000 × diluted rabbit polyclonal anti - human factor xii antibody (# 233504 , calbiochem ) and after washing with 3000 × diluted peroxidase - conjugated swine anti - rabbit immunoglobulins ( swarpo , # p0399 , dakocytomation , glostrup , denmark ). tht fluorescence of β 2 gpi was measured as follows . purified β 2 gpi from human plasma ( 400 μg ml − 1 final concentration ) was incubated with or without 100 μm cardiolipin ( cl ) vesicles or 250 μg ml − 1 of the factor xii activator dextran sulphate 500 k ( dxs500k , pharmacia , uppsala , sweden ), in 25 mm tris - hcl , 150 mm nacl , ph 7 . 3 . cl vesicles were prepared according to an established procedure . briefly , cl was dried under a stream of nitrogen . the lipids were resuspended to a concentration of 10 mg ml − 1 in 25 mm tris - hcl , ph 7 . 3 , 150 mm nacl by vigorous agitation , using a vortex . in the tht fluorescence assay , fluorescence of β 2 gpi in buffer , of cl or dxs500k in buffer , of buffer and tht alone , and of β 2 gpi - cl adducts and β 2 gpi - dxs500k adducts , with or without tht , was recorded as described above ( section tht fluorescence ). in addition , tem images were recorded with cl , β2gpi from human plasma , with or without cl , and with recombinant β2gpi , as described . 5 preparation of amyloid - like ovalbumin , human glucagon , etanercept and murine serum albumin to prepare structurally altered ovalbumin ( ova ) with amyloid cross - β structures , purified ova ( sigma , a - 7641 , lot 071k7094 ) was heated to 85 ° c . one mg ml − 1 ova in 67 mm nap i buffer ph 7 . 0 , 100 mm nacl , was heated for two cycles in pcr cups in a ptc - 200 thermal cycler ( mj research , inc ., waltham , mass ., usa ). in each cycle , ova was heated from 30 to 85 ° c . at a rate of 5 ° c ./ minute . native ova ( nova ) and heat - denatured ova ( dova ) were tested in the tht fluorescence assay and in the plg - activation assay . in the fluorescence assay and in the plg - activation assay , 25 and 100 μg ml − 1 nova and dova were tested , respectively . tem images of nova and dova were taken to check for the presence of large aggregates . modified murine serum albumin ( msa ) was obtained by reducing and alkylation . msa (# 126674 , calbiochem ) was dissolved in 8 m urea , 100 mm tris - hcl ph 8 . 2 , at 10 mg ml − 1 final concentration . dithiothreitol ( dtt ) was added to a final concentration of 10 mm . air was replaced by n 2 and the solution was incubated for two hours at room temperature . then , the solution was transferred to ice and iodoacetamide was added from a 1 m stock to a final concentration of 20 mm . after a 15 - minute incubation on ice , reduced - alkylated msa ( alkyl - msa ) was diluted to 1 mg ml − 1 by adding h 2 o . alkyl - msa was dialyzed against h 2 o before use . native msa ( nmsa ) and alkyl - msa were tested in the tht fluorescence assay and in the plg - activation assay . in the tht - fluorescence assay 25 μg ml − 1 nmsa and alkyl - msa were tested , and in the plg - activation assay 100 μg ml − 1 was tested . the presence of aggregates or fibrils was analyzed using tem . amyloid - like properties in human glucagon ( glucagen , # pw60126 , novo nordisk , copenhagen , denmark ) were introduced using a modified protocol based on the method described by onoue et al . 23 lyophilized sterile glucagon was dissolved at 1 mg ml − 1 in h 2 o with 10 mm hcl . the solution was subsequently kept at 37 ° c . for 24 hours , at 4 ° c . for 14 days and again at 37 ° c . for nine days . tht fluorescence was determined as described above , and compared with freshly dissolved glucagon . tpa - activating properties of both heat - denatured glucagon and freshly dissolved glucagon was tested at 50 μg ml − 1 . tem analysis was performed to assess the presence of large multimeric structures . protein assemblies with cross - β structure bind to immobilized fibronectin type i domains in a biacore surface plasmon resonance set - up we used a surface plasmon resonance set - up of biacore to test whether immobilized proteins with affinity for cross - β structure can capture amyloid - like polypeptides from solution under flow . this set up also allows testing of suitable elution buffers to disrupt the interaction . in this way insight into suitable methods to deplete proteins with cross - β structures from solutions is obtained , as well as insight into how to compete for the interaction of cross - β structure binders , which are , for example , immobilized on beads in a column , with proteins comprising cross - β structures . on one chip , we immobilized srage , tpa and k2p - tpa . one channel was left empty for reference purposes . protein solutions were centrifuged for 10 minutes at 16 , 000 * g before the solutions were applied to the biacore chip . centrifugation had no effect on the stimulatory effect of hb - age and amyloid β - globulins on tpa - mediated activation of plg ( fig1 , panel a ). moreover , we filtrated all protein solutions before they were applied to the biacore to exclude the presence of large aggregates with a density equal to buffer . for hb - age similar response units were obtained after centrifugation or filtration ( not shown ). subsequent experiments showed that hb - age , albumin - age and amyloid γ - globulins bind to immobilized tpa and srage ( fig1 , panels b - d ). the interaction of tpa and srage with hb - age and albumin - age could be disrupted with 0 . 1 m h 3 po 4 buffer ph 1 . 0 . amyloid γ - globulins , however , were not removed by this buffer . after trying several more harsh regeneration buffers , the binding capacity of the chip was lost . on a second chip , chemically synthesized hgfa f and tpa f , and fn f4 - 5 - his expressed in s . cerevisiae were immobilized . none of the polypeptides with cross - β structures bound to the two single f domain constructs . hb - age , endostatin and recombinant β2gpi bound , however , to the fn f4 - 5 doublet , whereas native hb did hardly bind ( fig1 , panels e - h ). affinities of the three proteins for fn f4 - 5 , expressed as the concentration of ligand that results in half maximum binding , ranges from 8 nm for hb - age , via 165 nm for recombinant β2gpi to up to 800 nm for endostatin . in fact , based on the absence of tpa activating properties in 100 μg ml − 1 endostatin ( fig1 , panel a ), we did not expect any binding at all . putatively , the surface plasmon resonance is more sensitive for the cross - β structure under the conditions used . we observed that when a stock solution of endostatin at 7 . 9 mg ml − 1 in the buffer as supplied by the manufacturer , is kept at ice or at room temperature , readily aggregates . perhaps , during the course of our experiments , part of the endostatin molecules start to denature , giving rise to the observed binding to fn f4 - 5 . with this chip , interaction between fn f4 - 5 and the protein ligands could be abolished simply by increasing the nacl concentration from 140 mm to 1 m . this shows that the interaction was primarily based on charge interactions . our surface plasmon resonance data show that f domains expressed in s . cerevisiae can bind to polypeptides with a cross - β structure . furthermore , the data show that both 0 . 1 m h 3 po 4 buffer ph 1 . 0 and 10 mm hepes ph 7 . 4 , 1 m nacl , 1 . 5 mm cacl 2 , 10 mm εaca , 0 . 05 % tween - 20 are suitable buffers to release polypeptides with a cross - β structure from cross - β structure - binding compounds . these buffers are also suitable to release cross - β structure - binding compounds and proteins that are bound to a ligand with a cross - β structure . these data are helpful during the design of a method to deplete solutions from cross - β structure rich compounds by using cross - β structure - binding polypeptides that are immobilized on a suitable supporting material . we analyzed a series of protein solutions that are used as therapeutics for human use for the presence of cross - β structures in the protein . protein solutions were stored at − 20 ° c ., 4 ° c . ( as recommended by the manufacturers ), room temperature , 37 ° c . or 65 ° c . fluorescence of congo red and tht in the presence or absence of the proteins was analyzed , as well as tpa binding , tpa activation and factor xii activation . for fluorescence assays , 10 μg ml − 1 aβ ( 1 - 40 ) e22q amyloid was used as a positive control and gave typical values of approximately 1250 and 1800 a . u ., respectively in the congo red - and tht fluorescence assay . furthermore , tem images were recorded to get insight whether amorphous aggregates are formed or fibrillar like structures . gelatin , cealb , fviii and to some extent gh , stored at the recommended storage temperature of 4 ° c ., enhanced the fluorescence of congo red ( fig2 , panel a ). in addition , cealb , gh and fviii enhance fluorescence of tht ( fig2 , panel b ). gh also induced tpa activation ( fig2 , panel c ). insulin activated tpa to a lesser extent , but still significantly ( fig2 , panel c ). both insulin and zinc - chelated insulin activate the factor xii / prekallikrein contact system ( fig2 , panel d ). gelatinous collagen fragments stored at 4 ° c . and 37 ° c . displayed enhanced congo red fluorescence in a storage temperature dependent manner ( fig2 , panel e ). only gelatin kept at 37 ° c . activated factor xii ( fig2 , panel f ). in an elisa set - up , binding of tpa was established for cealb , reopro , gelatin , zinc - chelated insulin ( fig2 , panel g ) and gh ( fig2 , panel h ), all stored at the recommended temperature of 4 ° c . for both elisas , hb - age was coated as a positive control ( not shown for clarity ). in the elisa depicted in fig2 , panel g , truncated k2p - tpa , or reteplase , which lacks the amyloid - binding f domain , was also tested for binding to the immobilized protein therapeutics . k2p - tpa did not bind to any of the therapeutics tested ( not shown ). on tem images various condensed aggregates are seen with modified gelatin ( fig2 , panel i ). gh appeared on tem images as linear , branched and condense particles , all apparently composed of spherical particles ( fig2 , panel j ). zinc - chelated insulin appears on tem images as thin linear unbranched fibrils with varying length ( fig2 , panel k ). fviii and reopro did not appear as visible particles under the electron microscope . cealb and insulin appeared as visible aggregates with no sign of a fibrillar nature ( fig2 , panels l , m ). reopro displayed storage temperature dependent tht fluorescence enhancement properties and tpa activating properties ( fig2 , panels n , o ). only after storage at 65 ° c . reopro enhanced tht fluorescence and induced pls activity . apparently , only at 65 ° c . reopro adopts the amyloid - like cross - β structure conformation . a tem image of reopro that was stored at the recommended temperature of 4 ° c . revealed that some non - fibrillar aggregates were present , that apparently do not have tht fluorescence enhancing or tpa activating properties under the conditions tested . based on the observed binding of congo red , tht and tpa , based on the appearance on tem images , and based on the observed activating properties towards tpa and factor xii , the tested solutions of cealb , gelatin , insulin , zinc - insulin , gh , reopro and fviii displayed amyloid - like properties , when stored under recommended conditions . for cealb , binding of tpa , congo red and tht is indicative for the presence of a cross - β structure . binding of congo red and activation of factor xii indicate the presence of cross - β structure conformation in gelatin . binding of tht and tpa , and activation of tpa by gh are indicative for amyloid - like properties in this solution . finally , both activation of tpa and factor xii by insulin / zinc - insulin are indicative for the presence of cross - β structure rich aggregates . these data show the presence of protein or peptide aggregates with amyloid - like properties or the potential that the cross - β structure can be formed upon storage in these protein solutions . structural analysis of the tested proteins can be expanded using techniques and assays such as x - ray diffraction experiments , fourier transform infrared spectroscopy , size exclusion hplc , cd spectropolarimetry and binding assays using amyloid binding proteins , and can be expanded by introducing new protein solutions in the series of analyses . factor xii and tpa bind to recombinant β 2 gpi and to β 2 gpi purified from frozen plasma , but not to β 2 gpi purified from fresh plasma recombinant β 2 gpi , but not β 2 gpi purified from fresh plasma stimulate tpa - mediated conversion of plg to pls , as measured as the conversion of the pls - specific chromogenic substrate s - 2251 ( fig3 , panel a ). an elisa demonstrated that tpa and factor xii bind recombinant β 2 gpi , but not to β 2 gpi purified from fresh human plasma ( fig3 , panels b , c ). recombinant β 2 gpi binds to factor xii with a k d of 20 nm ( fig3 , panel c ) and to tpa with a k d of 51 nm ( fig3 , panel b ). in addition , factor xii co - elutes from the anti - β 2 gpi antibody affinity column with β 2 gpi , which was purified from plasma that was frozen at − 20 ° c . and subsequently thawed , as shown on western blot after incubation of the blot with anti - factor xii antibody ( fig3 , panel d ). this shows that β 2 gpi refolds into a conformation containing cross - β structures upon freezing . fig3 , panel f shows that exposure of β 2 gpi to cl or dxs500k introduces an increased tht fluorescence signal , indicative for a conformational change in β 2 gpi accompanied with the formation of cross - β structure conformation . again , recombinant β 2 gpi initially already gave a higher tht fluorescence signal than native β 2 gpi purified from plasma . these data not only show that recombinant β2gpi already comprises more cross - β structure conformation than plasma β2gpi , but that recombinant β2gpi also adopts more readily this conformation when environmental factors change . in fig3 , panel g it is shown that exposure of β 2 gpi to cl , immobilized on the wells of an elisa plate , renders β 2 gpi with tpa binding capacity . binding of β 2 gpi directly to the elisa plate results in less tpa binding . these observations also show that cl has a denaturing effect , thereby inducing amyloid - like conformation in β 2 gpi , necessary for tpa binding . these observations , together with the observation that exposure of β 2 gpi to cl vesicles induced tht binding capacity ( fig3 , panel f ), show that exposure of β 2 gpi to a denaturing surface induces formation of amyloid - like cross - β structure conformation . furthermore , large fibrillar structures are seen on tem images of plasma β 2 gpi in contact with cl ( fig3 , panel g , image 2 and 3 ). small cl vesicles seem to be attached to the fibrillar β2gpi . images of plasma β 2 gpi alone ( fig3 , panel g , image 1 ) or cl alone ( not shown ) revealed that no visible ultrastructures are present . in contrast , non - fibrillar aggregates and relatively thin curly fibrils can be seen on images of recombinant β 2 gpi ( fig7 , panel g , image 4 ). these observation show that exposure of β 2 gpi to cl and expression and purification of recombinant β 2 gpi result in an altered multimeric structure of β 2 gpi , when compared to the monomeric structure observed with x - ray crystallography . 24 exposure of β 2 gpi to cl or dxs500k induces an increased fluorescence when tht is added , indicative for the formation of cross - β structure conformation when β 2 gpi contacts a negatively charged surface . we predict that the cross - β structure can be relatively easily formed by one or more of the five domains of the extended β 2 gpi molecule . 24 each domain comprises at least one β - sheet that may function as a seed for local refolding into a cross - β structure . in conclusion , it is revealed that several physical / chemical conditions influence the fold of the protein . exposure to cl or dxs500 , a freeze - thaw cycle , variations in protein purification protocol , the source of the protein and exposure to plastic all introduce a structural rearrangement in the protein accompanied by the formation of the amyloid - like cross - β structure fold . this new fold can be detected by , amongst others , tpa binding , tpa activation , factor xii binding and by conventional amyloid fluorescence assays . ova with amyloid - like properties was obtained by heat denaturation at 85 ° c . ( fig4 , panels a , b , i , k ). the presence of cross - β structures was established with tht fluorescence and plg - activation assays and by tem imaging . the fibrillar structures of at least up to 2 μm in length , seen on the tem images are likely not the only ova assemblies with cross - β structures present , as concluded from the observation that filtration through a 0 . 2 μm filter does not reduce the enhancement of tht fluorescence . a person skilled in the art can perform similar experiments with murine serum albumin ( msa ), human glucagon or etanercept , such as those described below ( fig4 ). the amyloid - like protein fold was induced in msa by heat denaturation at 85 ° c . and by reduction and alkylation of disulphide bonds ( fig4 , panels a - d ). we observed that also native msa enhanced tht fluorescence to some extent , but this was not reflected by stimulation of tpa activation . although heat - denatured msa and alkylated msa enhance tht fluorescence to a similar extent , they differ in tpa activating potential . this suggests that tpa and tht interact with distinct aspects of the cross - β structure . previously , we observed that congo red , another amyloid - specific dye , can efficiently compete for tpa binding to amyloid - like aggregates in elisas , whereas tht did not inhibit tpa binding at all ( patent application p57716ep00 and b . bouma , unpublished data ). amyloid - like cross - β structure conformation was induced in glucagon by heat - denaturation at 37 ° c . at low ph in hcl buffer ( fig4 , panels e , f , j ). in this way , a potent activator of tpa was obtained , that enhanced tht fluorescence to a large extent . in addition , long and bended unbranched fibrils were formed , as visualized on tem images ( fig4 , panel j ). noteworthy , at high glucagon concentration , also native glucagon had some tpa activating potential , indicative for the presence of a certain amount of cross - β structure rich protein . alkylated etanercept did not activate tpa at all , whereas heat - denatured etanercept had similar tpa activating potential as amyloid γ - globulins ( fig4 , panel g ). after heat denaturation , etanercept also efficiently induced enhanced tht fluorescence ( fig4 , panel h ). native etanercept both induced some tpa activation and gave some tht fluorescence enhancement . from our analyses we conclude that dova , alkyl - msa , heat / acid - denatured glucagon and heat - denatured etanercept comprise the cross - β structure conformation . the presence of the cross - β structures can be further established by circular dichroism spectropolarimetry analyzes , x - ray fiber diffraction experiments , fourier transform infrared spectroscopy , congo red fluorescence / birefringence , tpa binding , factor xii activation and binding , and more . 1 . cleland j . l ., m . f . powell and s . j . shire . the development of stable protein formulations : a close look at protein aggregation , deamidation , and oxidation . crit rev . ther . drug carrier syst . 10 , 307 - 377 ( 1993 ). 2 . wang w . protein aggregation and its inhibition in biopharmaceutics . int . j pharm . 289 , 1 - 30 ( 2005 ). 3 . krishnamurthy r . and m . c . manning . the stability factor : importance in formulation development . curr . pharm . biotechnol . 3 , 361 - 371 ( 2002 ). 4 . hermeling s ., d . j . crommelin , h . schellekens and w . jiskoot . structure - immunogenicity relationships of therapeutic proteins . pharm . res . 21 , 897 - 903 ( 2004 ). 5 . bouma b . et al . glycation induces formation of amyloid cross - beta structure in albumin . j . biol . chem . 278 , 41810 - 41819 ( 2003 ). 6 . nilsson m . r . techniques to study amyloid fibril formation in vitro . methods 34 , 151 - 160 ( 2004 ). 7 . bucciantini m . et al . inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases . nature 416 , 507 - 511 ( 2002 ). 8 . cribbs d . h ., b . y . azizeh , c . w . cotman and f . m . laferla . fibril formation and neurotoxicity by a herpes simplex virus glycoprotein b fragment with homology to the alzheimer &# 39 ; s a beta peptide . biochemistry 39 , 5988 - 5994 ( 2000 ). 9 . kayed r . et al . common structure of soluble amyloid oligomers implies common mechanism of pathogenesis . science 300 , 486 - 489 ( 2003 ). 10 . kranenburg o . et al . recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro . febs lett . 539 , 149 - 155 ( 2003 ). 11 . tucker h . m ., m . kihiko - ehmann , s . wright , r . e . rydel and s . estus . tissue plasminogen activator requires plasminogen to modulate amyloid - beta neurotoxicity and deposition . j . neurochem . 75 , 2172 - 2177 ( 2000 ). 12 . klein w . l ., g . a . krafft , and c . e . finch . targeting small abeta oligomers : the solution to an alzheimer &# 39 ; s disease conundrum ? 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