Patent Application: US-57487704-A

Abstract:
a protein must fold into a specific conformation in order to carry out its intended function . failure to fold correctly may lead to self - association and aggregation , and formation of a highly ordered and insoluble form of protein — the amyloid fibril . amyloid fibrils are associated with a growing number of human diseases and are thus of significant medical interest . current interest in amyloid has also grown within the wider protein science research community , based on the discovery that these unusual structures are likely to represent a generic form of all proteins . the invention provides amyloid fibrils , for novel biomaterials development , from a heterogeneous protein mixture . in particular it provides a protein derived as a secondary product from an industrial production process to make amyloid fibrils for novel biomaterials .

Description:
the inventors &# 39 ; research was aimed at producing amyloid - based materials from an accessible source of protein . to do this , research was directed to the factors that influenced amyloid formation . reliable protocols were established for the production of amyloid in a model system and then the protocols were experimented with using a protein source suitable for use in an industrial production scale . wheat flour proteins have conventionally been characterised on the basis of solubility ( osborne 1907 ), into the following groups : albumins and globulins ( soluble in dilute salt ), gliadins ( soluble in aqueous ethanol ), and glutenins ( soluble in dilute acid ). these groups are outlined in table 1 . the glutenins can be classified further into high molecular weight ( hmw ) and low molecular weight ( lmw ) categories . the albumins and globulins represent the non - gluten fraction of wheat protein , and the gliadins and glutenins the gluten fraction . the ability to isolate wheat protein into fractions of varying levels of purity allowed the authors to investigate the influence of protein purity in the wheat protein system , from heterogeneous mixtures of glutenins right down to solutions containing a glutenin subunit encoded by a single gene locus . for this initial research . the glutenin fraction was separated into protein components that were soluble in sds - phosphate buffer , and protein components that were not , using the extraction methods described by gerrard et al . ( 2001 ), based on the protocols of osborne ( 1907 ). the protein groups that did not solubilise after the initial buffer treatment were sonicated briefly to increase their solubility . the addition of a reducing agent was necessary to aid the separation of the hmw - and lmw - glutenin subunits ( egorov et al . 1998 ). the isolated glu - 1a gene locus encoded hmw - glutenin subunit 1 and glu - 1d gene locus encoded hmw - glutenin subunit pair 5 + 10 were supplied by k . sutton , crop and food research , and sourced from commercial wheat varieties using the methods outlined by sutton ( sutton 1991 ). attempts were made to induce the formation of amyloid from protein fractions extracted from wheat flour . it is well known to those practised in the art that the fractionation protocols presented herein represent the most commonly used procedures for wheat proteins and that alternative fractionation schemes ( e . g . use of iso - propanol or acetic acid ) will also yield similar heterogeneous protein mixtures that could be used as starting materials for amyloid formation . unless otherwise stated , all chemicals and reagents were purchased from sigma chemical company ltd ., aldrich chemicals or bdh laboratory supplies , and were of analytical grade . the wheat derived protein used in the described extractions was either purchased from a local supermarket , or milled and supplied by crop and food research . the flour sources were of varying levels of quality and composition . the hmw glutenin subunits were extracted and alkylated using the method of sutton ( 1991 ). the a - gliadin fraction was supplied by crop and food research , and extracted using in - house techniques . ph measurements were obtained using an ultrabasic ub10 ph meter purchased from denver instrument co . and fitted with a high - performance glass body ph / atc electrode . the electrode was calibrated daily against standard buffers at ph 4 . 0 , 7 . 0 and 9 . 0 , purchased from bdh laboratory supplies . polyacrylamide gel electrophoresis was routinely run using a bio - rad mini protean ® cell kit powered by a bio - rad 300 power pack , and gradipore precast 8 - 16 % acrylamide gradient igels . ultraviolet ( uv ) spectroscopy was performed using a hewlett packard 8452a diode array spectrophotometer interfaced with a personal computer running hewlett packard 8452a uv - visible operating software . fluorescence spectroscopy was performed using a cary eclipse varian fluorescence spectrophotometer interfaced with cary eclipse version 2 operating software . microscopy imaging was carried out using a j . swift & amp ; son optical microscope fitted with a cross - polarising lens . electron micrographs were obtained using a joel jem - 1200 ex transmission electron microscope operating at 80 kv . wheat flour was separated into four protein fractions using the methods of gerrard et al . ( 2001 ), based on the protocols of osborne ( osborne 1907 ). this sequential four step extraction procedure was carried out on a scale of between 100 mg to 5 g of wheat flour . firstly , albumins and globulins were extracted using a 2 % ( w / v ) sodium chloride solution ( 0 . 4 ml per 100 mg of wheat flour ). the mixture was pulse vortex mixed every 5 min for 30 min , then centrifuged at 10 , 000 g for 5 min . the clarified supernatant containing albumins and globulins was stored at − 20 ° c . the resultant pellet was resuspended in 70 % ( v / v ) aqueous ethanol solution ( 0 . 4 ml per 100 mg of initial wheat flour weight ) to extract the gliadin protein fraction . this mixture was pulse vortex mixed every 5 min for 30 min , then centrifuged at 10 , 000 g for 5 min . the clarified supernatant containing gliadins was stored at − 20 ° c . the solid fraction was re - extracted in sds - buffer ( 0 . 5 % ( w / v ) sds , 0 . 05 m phosphate , ph 6 . 9 , 0 . 4 ml per 100 mg of initial wheat flour weight ) to separate the sds - soluble glutenin protein fraction . the mixture was pulse vortex mixed every 5 min for 30 min , then centrifuged at 10 , 000 g for 5 min . the resultant clarified supernatant was stored at − 20 ° c . finally , the residual pellet was extracted with a sds - buffer ( 0 . 5 % ( w / v ) sds , 0 . 05 m phosphate , ph 6 . 9 , 0 . 4 ml per 100 mg of initial wheat flour weight ). the heterogeneous mixture was sonicated at . 30 w for 15 sec using a branston model 250 sonic disruptor . after vortexing , the samples were centrifuged at 10 , 000 g for 5 min . the clarified sds - insoluble glutenin supernatant was stored at − 20 ° c . each protein fraction was freeze dried and restored at − 20 ° c . wheat flour samples were extracted on a 100 mg scale . the total glutenin fraction of the wheat flour was extracted using a propan - 1 - ol / dithiothreitol ( dtt ) mixture ( 0 . 625 ml of 50 % ( v / v ) propan - 1 - ol , 1 % ( w / v ) dtt ) heated at 60 ° c . for 30 min with pulse vortex every 5 min . the samples were centrifuged at 20 , 000 g for 10 min . an aliquot ( 0 . 5 ml ) of the supernatant was used in the subsequent procedure . the concentration of propan - 1 - ol was then adjusted to 60 % ( v / v ). the mixture was pulse vortexed and allowed to stand at room temperature for 30 min . the precipitated hmw - glutenin subunits were isolated by centrifuging at 3000 g for 5 min and were washed three times with 60 % ( v / v ) propan - 1 - ol ( 1 ml ). the hmw - glutenins were dissolved in reducing buffer ( 0 . 5 ml , 0 . 08m tris - hcl , ph 7 . 5 , 50 % propan - 1 - ol ( v / v ) 1 % dithiothreitol ( dtt ), ( w / v )) heated at 60 ° c . for 30 min , with agitation every 10 min . the resulting reaction mixture was allowed to cool to room temperature before addition of the alkylating buffer ( 0 . 5 ml , 0 . 08 m tris - hcl , ph 7 . 5 , 50 % propan - 1 - ol ( v / v ) 3 % 4 - vinylpyridine ( v / v )). care was required with this step as the 4 - vinylpyridine causes severe burns . the alkylation reaction mixture was heated in a water bath at 60 ° c . for 15 min . after cooling to room temperature , this solution was acidified by the addition of glacial acetic acid ( 0 . 1 ml ). salts were removed by dialysing in 0 . 01 m acetic acid solution overnight . finally the solvent was removed by freeze drying to yield alkyated hmw - glutenin subunits that were soluble in weak acids . all extracted wheat flour protein fractions were resuspended in solutions at the concentrations given in the table below ( table 2 ). temperatures of 25 ° c .- 37 ° c . addition of acids ( h 2 so 4 , hcl , various concentrations including 0 . 5 m ) addition of organic solvents ( 30 % trifluoroethanol , 80 % ethanol ) addition of reducing compounds ( mercaptoethanol , various concentrations including 1 m ) addition of denaturants ( urea , various concentrations including 2 m ) addition of preformed insulin ‘ seeding ’ molecules ( 10 - 20 μl of insulin amyloid , prepared fresh using the flash - freeze protocol of klunk et al . ( 1989b )). the appropriate reagents were added to the reaction mixtures before incubation . all treatments were prepared in triplicate . for each treatment system samples frozen without heating were used as controls . aliquots ( 120 μl ) were removed at approximately 10 day intervals for a period of up to 70 days . these aliquots were frozen at − 20 ° c . until required for analysis by a combination of tht assay , cr assay and tem imaging . protein concentration in the resuspended solutions was determined using a modified version of the bradford method ( 1976 ). a standard curve with bovine serum albumin ( bsa ) or insulin was determined daily . the absorbance is linear for concentrations from 1 to 15 μg / ml of bsa . the protein concentration of unknown solutions was determined by the addition of bio - rad bradford reagent ( 200 μl ) to solution ( 800 μl ). if the absorbance reading was outside the linear range of the standard curve it was necessary to dilute ( or concentrate ) the sample to ensure accuracy of the results . after mixing and incubation at room temperature for 6 min , the absorbance of each sample was measured at 595 mm against a distilled water blank . all analyses were performed in triplicate . each sample was placed in a labelled eppendorf tube and resusupended in a loading buffer ( 0 . 5 m tris - hcl buffer ph 6 . 8 , 5 % sds ( w / v ), 20 % glycerol ( v / v ), 10 % 2 - β - mercaptoethanol ( v / v ), 0 . 5 % bromophenol ( w / v )). the samples were placed in a boiling water bath for 2 min to help solubilise the samples and to aid the reduction of disulfide bonds . protein samples were run against wide range molecular weight markers purchased from sigma ( 6 , 500 - 205 , 000 da ). each boiled protein sample and marker was loaded into separate wells in the polyacrylamide gel . samples were electrophoresed at a constant voltage of 150 v in running buffer ( 20 mm tris , 20 mm glycine , 0 . 5 % sds w / v ph 8 . 3 ) at 4 ° c . the proteins in the resultant polyacrylamide gel were stained in a solution of coomassie brilliant blue stain ( 0 . 1 % coomassie brilliant blue ( 10 % v / v ), glacial acetic acid ( 50 % v / v ), methanol , dh 2 o ( 40 % v / v ), pre - filtered at room temperature ). after two hours of staining the gel was transferred into a destaining solution ( glacial acetic acid ( 10 % v / v ), methanol ( 5 % v / v ), dh 2 o ( 85 % v / v )) at room temperature . the protein samples were analysed using a range of analytical techniques developed specifically for the detection of amyloid material . samples could be stained using the dye congo red ( westermark et al . 1999 ), or via the use of calorimetric ( klunk et al . 1989a , klunk et al . 1989b ) or fluorescence ( levine 1999 ) assays . samples were stained for amyloid following the procedure of westermark ( 1999 ). the protein samples were fixed onto glass slides pre - coated in poly - l - lysine , prepared by submerging in a solution of 0 . 1 % poly - l - lysine overnight , with drying at 337 ° c . to stain the samples , the slides were soaked in prefiltered working solution a ( 170 mm nacl , 80 % ethanol ( v / v ), naoh ( 0 . 001 %, added prior to filtering )) for 20 min . slides were then transferred directly to a prefiltered working solution b ( 0 . 2 % congo red ( w / v ), 170 mm nacl , 80 % ethanol ( v / v ), naoh ( 0 . 001 %, added prior to filtering )) and soaked for a further 20 min . this was followed by two brief washes in 100 % ethanol . using this method , samples appeared as red - orange material under a light microscope . when viewed under cross - polarised light , where one light filter is rotated at 90 ° with respect to a second light filter , a bright green or ‘ apple green ’ birefingence could be observed in areas where amyloid was present . the method for this binding assay involves a modification of the protocol described by klunk et al . ( 1989 ). the spectral measurements were taken using a hp - 8452a diode array spectrophotometer set in the wavelength - scanning mode to read from 300 to 700 nm . to analyse the wheat protein fractions , a solution of congo red ( 1 mm ) was prepared in a hepes buffer ( 0 . 01 m hepes , 2 . 7 mm kcl , 0 . 137 m nacl , ph 7 . 4 ), containing 10 % ethanol to prevent the formation of micelles ( micro - aggregation ). this solution was filtered three times through gelman extra - thick glass fibre filters before use , and stored at room temperature protected from light . the protein concentration of the samples to be tested was between 10 - 20 μm . all samples were assayed in duplicate . congo red solution was diluted with phosphate buffer immediately prior to use in the spectrophotometric assays , typically to give a final congo red concentration of 10 μm in the protein sample solution to be analysed . congo red solution and protein solution were mixed in 1 ml cuvette and allowed to stand at room temperature for 30 min before spectral analysis . the spectrum of the resulting mixture was measured between 300 - 700 nm . the spectra of buffer , buffer with congo red and buffer with protein were the experimental controls . a shift of the spectrum to longer wavelengths and a new point of maximal difference at 540 nm is evidence for the presence of amyloid ( klunk et al . 1999 ). the thioflavin t ( tht ) method used throughout this research was based on the protocols of le vine ( 11999 ). the wavelength of excitation was 450 nm . the complete emission spectra were recorded , over a range that included the wavelength of 482 mm . both excitation and emission slits were set to 5 nm . in the cuvette , protein sample was added to a tris buffer ( 50 mm tris , 100 mm nacl , ph 7 . 5 ) containing tht ( 5 to 10 μm ). the final concentration of protein in the cuvette was typically 20 μg / ml . the resulting solution was mixed and left to stand at room temperature for 3 min to allow binding between the dye and protein to equilibrate , before the emission spectra were recorded . the spectra of buffer only , buffer with tht and buffer with protein were the experimental controls . emission spectra for all samples were assayed in duplicate . the experiments were also conducted in well plates that were standardised as for the cuvette procedure . the wheat protein concentration for these assays was approximately 0 . 5 mg / ml . the final volume in the well plates was 200 μl . the protein samples were analysed by tem based on the protocols described by whittingham et al . ( 2002 ) and burke and rougvie ( 1972 ). tem samples were prepared by placing small aliquots ( 5 ml ) of protein fibril samples on carbon - coated copper grids . grids were then rinsed with distilled water , negatively stained with 1 % uranyl acetate , and dried before imaging commenced . the general protocols used can be found above . additional specific details are described below : the wheat flour was separated into fractions using the protocols outlined in methods 2 ( wheat protein extraction ) and 3 ( hmw - glutenin extraction and alkylation procedure ), using initially both flour purchased from the supermarket ( extractions for gliadin and glutenin fractions ) and flour supplied by crop and food research ( as above but also for hmw - glutenin subunits ). flours from specific wheat cultivars were used to extract the hmw - glutenins but these did not appear to form fibrils ( glu - 1a gene locus encoded subunit 1 , and glu1d gene locus encoded subunit pair 5 + 10 ). the supernatants containing the solubilised protein fractions were pooled and lyophilized to concentrate the protein content . incubations were carried out involving protein fractions that had not been lyophilised and hence were less concentrated ( i . e . & lt ;& lt ; 10 mg / ml ). these samples did not conclusively form fibrils . however , based on the successful results with the higher concentration ( lyophilised ) samples , the possibility that these samples could have ( given more time ) can not be excluded . the lyophilised protein fractions were then redissolved in either the appropriate extraction buffer or nanopure water ( table 2 ), typically to give a final protein concentration of approximately 10 mg / ml . the protein samples tested ranged in final concentration from 6 . 5 - 15 mg / ml , but typically were 10 mg / ml . the majority of the protein samples were then used for the various incubation treatments with no further treatment , while a selection of the samples were dialysed against distilled water to remove sds and phosphate buffer salts . the samples were then placed in 1 . 5 ml eppendorf tubes and incubated at either 25 ° c . or 37 ° c . the volume of each tube was initially between 1 - 1 . 5 ml , with each sample condition prepared in triplicate . a separate volume of at least 120 μl was frozen at − 20 ° c . ( for glutenins ) or − 80 ° c . ( for gliadins , in the ethanol based solution ) as a time zero control . the invention provides a method for the formation of amyloid that can be used as a model to study amyloid assembly and structure and particularly to study the formation of amyloid in diseases such as alzheimer &# 39 ; s , parkinson &# 39 ; s and encephalopathies with a view to understanding how these diseases could be treated . the invention also provides a novel new source of amyloid protein , suitable for applications : in nanotechnology ( e . g . generation of silver or other metal nanowires ); as material scaffolds for tissue and bone growth ; as target - selective porous channels in films and membranes ( e . g . for bioremediation ); for strength enhancement of composites and films ; and as biosensors . bellotti . v . p . mangione , and m . stoppini . 1999 . biological activity and pathological implications of misfolded proteins . cellular and molecular life sciences 55 : 977 - 991 . bradford , m . a . 1976 . a rapid and sensitive method for the quantitation of microgram quantities of protein utilising the prinicle of protein dye binding . analytical biochemistry . 72 : 248 - 254 . burke , m . j ., and m . a . rougvie . 1972 . cross - ÿ protein structures . 1 . insulin fibrils . biochemistry 11 : 2435 - 2439 . chen . s ., v . berthelier , j . b . hamilton , b . o &# 39 ; 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