Patent Application: US-99586506-A

Abstract:
the present invention relates to agents comprising non - natural protein sequences with at least two protein or polypeptide epitopes that are relationally linked by a common or shared functional relationship such as being components in a series of components of the same metabolic or signal transduction pathway or a pathway associated with interaction between two systems such as host - parasite or host - pathogen . the invention further includes a method of simultaneously calibrating and investigating the quantitative relationships between the at least two protein or polypeptide epitopes .

Description:
taking , by way of example only a process a → b → c → d → e , catalysed by enzymes f , g , h , and i . antibodies specific to each enzyme exist ( called f ′, g ′, h ′, and i ′) and the epitope for each of these antibodies has been defined ( f ″, g ″, h ″, and i ″). a calibration standard material could be constructed in which the chemical constituent of each antibody epitope ( f ″, g ″, h ″, and i ″) is linked in series to form an unnatural protein sequence . this sequence is then linked in series to an additional protein sequence or scaffold portion , which is not recognised by any of the antibodies in this particular experimental series . this additional protein mass or scaffold portion functions in controlling the molecular weight of the non - natural protein sequence of the present invention . the non - natural protein sequence product will contain known amounts of each epitope , and can thus be used in experiments in known amounts to calibrate the signals generated by the experiment . the non - natural protein sequence of the present invention is shown schematically in fig1 , in which a series of 11 epitopes are linked in series to a scaffold portion also termed as an irrelevant protein or concatamer as in the figure . many proteins can be expressed in a variety of isoforms , either from the expression of closely related genes or from the production of alternatively spliced forms of an individual gene , or by combination of both of these mechanisms . the multifunctional serca sarcoplasmic / endoplasmic reticulum ( ca 2 + — mg 2 + )- adenosine triphosphoatase exists in a number of isoforms generated from different genes ( 1 , 2 , 3 ), with alternative splicing products of genes serca1 and serca2 resulting in further diversity . see table 1 below for details . with reference to fig2 , there is shown a schematic representation of a non - natural protein sequence of the present invention which can be used to investigate isoforms of serca sarcoplasmic / endoplasmic reticulum ( ca 2 + — mg 2 + )- adenosine triphosphoatase the multifunctional protein phosphatase , calcineurin ( can ) is an example of an enzyme expressed in a variety of isoforms . can is involved in a large variety of biological events including programmes of gene expression in response to extracellular signals ( can / nfat ). the calcineurin isoforms are as follows : calcineurin alpha ( can alpha ); calcineurin beta ( can beta ); and calcineurin gamma ( can gamma ) sequence not commercially available . a product of the invention comprises the three calcineurin isoforms and table 2 below for details . the multifunctional protein kinase , calmodulin - dependent kinase ii is an example of an enzyme expressed in a variety of isoforms . cam kinase ii is involved in a large variety of biological events including memory , regulation of vesicle movement , and maladaptive responses in heart failure . the isoforms of camkii are listed below : a calibration product could be constructed from a series of epitope sequences , where each sequence represents the epitope for an antibody specific for an isoform of cam kinase ii . some epitopes are shared between all or several isoforms , these epitopes could be incorporated in the calibration standard to calibrate multiple isoforms with a single antibody ( e . g . module 5 ). a number of phosphorylation sites exist in the protein . epitopes for phosphorylation site specific antibodies could be incorporated in the product ( e . g . module 6 above ) to calibrate the status of phosphorylation too . details of the antibodies and epitopes are set out below in table 3 . polymorphisms occur within biological species in probably every gene . in some cases the polymorphisms occur with altered probability in disease situations , and in those case are of particular interest and use . genetic variation exists within the population of a species , which at the individual gene level is manifest as polymorphisms of a gene . polymorphisms represent typically single base changes in the sequence of a gene which can occur in the coding or non - coding regions . these deviations in sequence can be without consequence to the gene , or can alter the level of expression of the gene , or can alter the polymer encoded by the gene . in many instances the probability of disease is linked to particular polymorphisms , which serves as a useful screening tool , and as a basis for hypothesis driven research into the cause and management of disease . certain polymorphisms in the ryr2 gene , which encodes an ion channel expressed in the heart , are associated with a disorder known as catecholaminergic polymorphic ventricular tachycardia ( cpvt ) which can provoke electrical irregularity and sudden death when an individual exercises . to date , over 20 separate mis - sense polymorphisms ( those which alter the primary sequence of the protein ) have been discovered in the human ryr2 gene , which are linked to cpvt . these include : which are residues conserved between man ( and mouse ), drosophila and caenorhabditis elegans . they exist in regions which are also highly conserved , both across these species , and between isoforms of ryr ( 1 , 2 , and 3 ). a large number of mutations in ryr2 are found in patients with arrhythmogenic right ventricular dysplasia type 2 ( arvd2 ) and catecholaminergic polymorphic ventricular tachycardia ( cpvt ). these are believed to play a causal role in disease . a sub - set of known disease associated mutations of ryr2 include : ( 1 ) r176 q , ( 2 ) v2306 i , ( 3 ) g3946 s and ( 4 ) v4653 f . to our knowledge antibodies specific for these mutations do not exist , however it is likely that they can be generated using short synthetic peptide immunogen incorporating the mutation site , using techniques known in the art . calibration of such antibodies could be achieved using a product comprising seq id nos 19 - 22 ( see table 4 below ). protein p53 ( also called tp53 ) is associated with a high proportion of cancers in man . for example in human liver cancer , 26 % of cases ( 559 of 2153 tumours ) show mutation in tp53 according to 64 studies ( jackson et al ; 2006 toxicology science 90 , 400 - 418 ). similarly tp53 mutations occur in 42 % of spontaneous lung tumours in man . missense polymorphisms result in mutant proteins , some of which are associated with cancer , such as : ( 1 ) r 249 s — most frequent tp53 mutation in hepatocellular carcinoria ( hcc ), ( 2 ) r172p , ( 3 ) r172h and ( 4 ) r270h . to our knowledge antibodies specific for these mutations do not exist , however it is likely that they can be generated using short synthetic peptide immunogen incorporating the mutation site , using techniques known in the art . calibration of such antibodies could be achieved using the following product comprising seq id nos 26 - 29 of the epitopes after mutation , see table 5 below : a calibration standard comprising epitopes for antibodies to each of these proteins would be useful in the study of glycolytic processes in biology , biotechnology and medicine . the eukaryotic cell cycle is an essential pathway necessary for all proliferative responses . this cycle involves a number of protein kinases and their partner regulatory proteins ( cyclins ), the concentration of the cyclins change throughout the cell cycle to allow passage of the cell through specific controlling check - points . cell cycle engine parts include : ( 1 ) cyclin a ; ( 2 ) cdk1 ; ( 3 ) cyclin d ; ( 4 ) cdk4 ; ( 5 ) cdk6 ; ( 6 ) cyclin e ; ( 7 ) cdk2 ; and ( 8 ) cyclin b . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 8 as defined in table 6 below would be useful in the study of cell cycles in biology , biotechnology and medicine . a series of components acting in consort can form a pathway . large numbers of pathways exist in biochemistry . one example is a pathway of interactions which control the expression of cell cycle regulators , cyclin a and cyclin e . g1 cyclins that overcome inhibitors of cell cycle progression are : ( 1 ) p16 ; ( 2 ) cyclin d ; ( 3 ) retinoblastoma protein ; ( 4 ) e2f ; ( 5 ) cyclin e ; ( 6 ) cyclin a ; and ( 7 ) p27 . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 7 as defined in table 7 below would be useful in the study of cell cycles in biology , biotechnology and medicine . an example of a pathway of interactions which communicate extracellular stimuli to changes in gene expression , involving nfat and calcineurin is : ( 1 ) chp ; ( 2 ) fk506 ; ( 3 ) mcip / calcipressin ; ( 4 ) akap79 ; ( 5 ) csa / cya ; ( 6 ) cabin 1 / cain ; ( 7 ) nfat ( p ); ( 8 ) pka ; ( 9 ) cki ; ( 10 ) gsk - 3beta ; ( 11 ) jnk ; ( 12 ) p38 ; ( 13 ) mef2 ; and ( 14 ) nfat . table 8 below shows the details for the manufacture a non - natural protein sequence of the present invention using two or more epitopes selected from the group comprising epitopes 1 - 14 . a further example pathway of interactions is control of the production of cytokines downstream of the toll - like receptor . lipopolysaccharide is a ligand for the toll - like receptor and the proteins involved in the signalling network include : ( 1 ) tlr ; ( 2 ) trif ; ( 3 ) traf6 ; ( 4 ) jnk ; ( 5 ) il10 ; ( 6 ) il6 ; ( 7 ) rantes ; ( 8 ) gcsf ; ( 9 ) tnf alpha ; and ( 10 ) mipi alpha . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 10 as defined in table 9 below would be useful in the study of cytokine production in biology , biotechnology and medicine . some biological components share a common location in a cell for all or some of their time . a number of signals contained within the primary sequence of proteins control their location in the cell . residence of this location is typically dynamic ( rather than static ) and thus evaluation of the entire protein complement of that location would be valuable in biological research . lipid raft domains of biological membranes are an interesting example of a discrete cellular location . our present understanding places lipid rafts as subdomains of the plasma membrane , characterised by a gel phase lipid composition ( lipid and cholesterol ) which allows residence of some particular proteins and exclusion of others . three distinct lipid raft types can be resolved , as summarised in table below ( taken from http :// www . bms . ed . ac . uk / research / others / smaciver / cyto - topics / lipid rafts and the cytoskeleton . htm ): the centrosome is a common physical location for some biological components . it is located adjacent to the eukaryotic nucleus and serves a variety of functions including the organisation of microtubules . the centrosome organises the assembly of the mitotic spindle which permits the correct segregation of chromosomes . abnormalities in centrosome components can lead to centrosome dysfunction , which is often associated with proliferative diseases such as cancer . centrosomes also play important roles in cell migration , the movement of cilia and the movement of vesicular membrane structures within cells . centrosome contains a number of proteins , including : ( 1 ) microtubule ; ( 2 ) pericentrin ; ( 3 ) centrin ; ( 4 ) pcmi ; ( 5 ) ninein ; ( 6 ) bbs4 ; ( 7 ) p150 glued ; ( 8 ) dynein ; ( 9 ) centriolin ; ( 10 ) gamma tubulin ; ( 11 ) polo kinases ; ( 12 ) aurora kinases ; ( 13 ) catanin ; and ( 14 ) katanin . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 14 as defined in table 10 below would be useful in the study of cell cycles in biology , biotechnology and medicine . furthermore a calibration product comprising amino acid sequences to antibodies specific to multiple components below is envisaged . in some instances suitable antibodies with known epitope sequences have been described , in other instances such antibodies need to be identified . lipid rafts is another physical location , which is a domain of the plasma membrane phase separated from surrounding regions of membrane . the phase separation arises as a consequence of the concentration of cholesterol and sphingomelin lipids , which group together to form a gel phase . transmembrane proteins typically cannot enter this microdomain , which is populated instead by proteins anchored through fatty acid , or lipid - like units , including : gpi ( glycosylinositolphosphatidyl ) anchored proteins and proteins which are both myristolated and palmitoylated . a series of proteins associated with lipid rafts include : ( 1 ) lck ( src kinase family members ); ( 2 ) fyn ( src kinase family members ); ( 3 ) h - ras ; ( 4 ) zap - 70 ; ( 5 ) cd3ç ; ( 6 ) lat ; ( 7 ) flotillin - 1 ; ( 8 ) cd2 ; ( 9 ) pag ; ( 10 ) f - actin ; and ( 11 ) cd59 . a calibration product comprising amino acid sequences to antibodies specific to multiple components above is conceivable . in some instances suitable antibodies with known epitope sequences have been described , in other instances such antibodies need to be identified . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 11 as defined in table 11 below would be useful in the study of lipid rafts in biology , biotechnology and medicine . macromolecular complexes which bring post - translational modification enzymes close to their target substrates are known . akaps ( a - kinase anchoring proteins ) are a good example of such complexes , which target enzymes involved in signalling with their substrate or effector proteins , creating local signalling circuits . the makap macromoleular complex contains : ( 1 ) de4d3 ; ( 2 ) makap ; ( 3 ) pka ; ( 4 ) epac1 ; ( 5 ) rap1 ; ( 6 ) mekk ; ( 7 ) mek5 ; and ( 8 ) erk5 . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 8 as defined in table 12 below would be useful in the study of lipid rafts in biology , biotechnology and medicine . many biological polymers assemble stably or transiently into macromolecular complexes , which typically exhibit function . one such complex is the dystrophin complex which forms a junction between the plasma membrane of a muscle cell and the underlying cytoskeletal structure . the dystrophin complex contains a number of proteins , including : ( 1 ) laminin ; ( 2 ) alpha dystroglycan ; ( 3 ) beta dystroglycan ; ( 4 ) caveolin ; ( 5 ) dystrobrevin ; ( 6 ) dystrophin ; ( 7 ) actin ; ( 8 ) alpha sacroglycan ; ( 9 ) beta sacroglycan ; ( 10 ) delta sacroglycan ; ( 11 ) gamma sacroglycan ; and ( 12 ) sacrospan . a product comprising the non - natural protein sequence comprising two or more epitopes selected from 1 - 12 as defined in table 13 below would be useful in the study of lipid rafts in biology , biotechnology and medicine . the cardiac ryanodine receptor ( ryr2 ), located in the sarcoplasmic reticulum ( sr ), is a calcium release channel which is centrally involved in the myocyte excitation - contraction ( e - c ). the ryanodine recpetor is also the center of a massive macromolecular complex , which includes numerous regulatory proteins that can modulate ryr2 function . this complex includes proteins that interact with the cytoplasmic part of the ryr2 directly or indirectly ( e . g . calmodulin ( cam ), fk - 506 - binding proteins , protein kinase a , ca - cam - dependent protein kinase , phosphatases 1 and 2a , makap , spinophilin , pr130 , sorcin , triadin , junctin , calsequestrin and horner ). understanding both the physical / molecular nature of the protein - protein interactions between ryr and these other proteins is important since this complex and the modulation of the ryanodine receptor is believed to be involved in cardiac arrhythmias , pace - maker function of the heart and cardiac disease . fig5 shows all of the known protein - protein interaction sites between ryr2 and the macromolecular complex components . the proteins labelled with red numbers are some of the proteins which are involved in the complex , and which have been used to produce a corresponding calibrant . antibody epitopes for the proteins in this complex have been genetically encoded and expressed in bacteria to produce a single protein ( fig6 ) that contains all of the antibody epitopes to each of the labelled proteins . the numerical notation of each protein corresponds to the notation in table 14 below . notations a and b can be cleaved and removed , which allows for the production of the true ryr2 macromolecular complex epitope calibrant . notations 1 and 10 are tags which can be used in purification of the calibrant protein and also act as common and widely used antibody epitopes . the optimised genetic sequence , which encodes for all of the proteins in table 14 is shown above as seq id no : 88 . below as seq id no : 89 is the resulting amino acid sequence from the genetic code the genetic sequence encoding the antibody epitopes that make up the calibrant ( and purification tags ) is synthesised and inserted into the e . coli expression vector pgs - 21a ( fig7 ). the pgs - 21a plasmid , which now contains the genetic sequence to encode for the ryr2 macromolecular complex calibrant , is transformed into bl21 ( de3 ) plyss e . coli cells . transformed cells are selected and used to express the calibrant protein after the induction of gene synthesis with iptg . after 3 . 5 hours of expression , the cells were harvested and re - suspended in sample buffer for analysis by sds - page and western blot . this was done in order to assess the purity of the calibrant product . fig8 shows a western blot result of a doubling dilution series of the expressed calibrant . following successful expression of a considerably pure calibrant product , the calibrant expression was scaled up from 1 ml to 1 . 5 l ( fig9 and 11 ) and was used to produce a quantifiable calibration plot ( fig1 and 12 ). the calibration curve was performed in triplicate and each replica was probed with a different epitope antibody and quantified by measuring the densitometry of the expressed ryr2macromolecular complex calibrant . mitogen activated protein kinases ( mapk ) are at the center of many signalling transduction pathways in eukaryotic cells . the study of mapk pathways is important in the research of many disease areas such as inflammation , cancer and parkinsons disease . we have used several proteins which are involved in one of the mapk pathways to produce a corresponding calibrant . antibody epitopes for the proteins on the in this pathway have been genetically encoded and expressed in bacteria to produce a single protein that contains all of the antibody epitopes to each of the labelled proteins . the numerical notation of each protein corresponds to the notation in table 15 below . notations a and b can be cleaved and removed , which allows for the production of the true mapkinase pathway epitope calibrant . notations 1 and 11 are tags which can be used in purification of the calibrant protein and also act as common and widely used antibody epitopes . the optimised genetic sequence , which encodes for all of the proteins in table 15 is shown below as seq id no : 100 . the resulting amino acid sequence from the genetic sequence of seq id no : 100 is given below as seq id no : 101 . the genetic sequence encoding the antibody epitopes that make up the calibrant ( and purification tags ) is synthesised and inserted into the e . coli expression vector pgs - 21a ( fig1 ). the pgs - 21a plasmid , which now contains the genetic sequence to encode for the mapk pathway calibrant , is transformed into bl21 ( de3 ) plyss e . coli cells . transformed cells are selected and used to express the calibrant protein after the induction of gene synthesis with iptg . after 3 . 5 hours of expression , the cells were harvested and re - suspended in sample buffer for analysis by sds - page and western blot . this was done in order to assess the purity of the calibrant product . fig1 shows a western blot result of a doubling dilution series of the expressed calibrant . following successful expression of a considerably pure calibrant product , the calibrant expression was scaled up from 1 ml to 1 . 5 l ( fig1 , 18 and 20 ) and was used to produce a quantifiable calibration plots ( fig1 , 19 and 21 ). the calibration curve was performed in triplicate and each replica was probed with a different epitope antibody and quantified by measuring the densitometry of the expressed mapkinase pathway calibrant . 1 . stamm s ., ben - 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