Patent Application: US-47053404-A

Abstract:
the present invention relates to a novel human polypolymerase enzyme , its nucleic and amino acid sequence and use thereof as well as an antibody against the novel enzyme and use thereof . the novel enzyme is named pap gamma and is not related to the previously known paps .

Description:
the novel papγ was cloned and recombinant proteins were expressed in a prokaryotic expression system . procedure for cloning and purification are found below . initial kinetic analysis revealed that the new human papγ is equally good or better compared to the human paps encoded by the papola gene . all nucleotide and amino acid numbers below refer to seq id no 3 and 4 , respectively , as these are the preferred ones . in fig1 it is shown that the recombinant papγ has high specificity for synthesing poly ( a ) tails , using the non - specific assay in the presence of mn ( ii ). the reaction mixture contains in final concentrations : 100 mm tris / hcl buffer ph 8 . 6 ( rt ), 40 mm kcl , 40 μm edta , 10 % glycerol , 1 . 0 mm dtt , 0 . 9 units rnasin ( ribonuclease inhibitor ), 0 . 1 % np - 40 , 0 . 5 mm mncl 2 , 0 . 5 mg / ml bsa and a mixture of cold and radiolabeled rna substrate oligoa ( 15 ) ( 330 fmoles ); pap is added to the reaction at 3 different amounts of 17 . 5 , 35 , and 70 ng , respectively . the reactions proceed at 30 min , 37 ° c . the reaction is stopped by proteinase k buffer , the rna is extracted by phenol : chloroform and it is run in a 16 % acrylamide : bis - acrylamide ( 19 : 1 )- 7 m urea gel . the gel is exposed overnight to a phosphoimager screen and further analyzed . the lanes represent the following : lane 1 : negative control ( no pap ), lanes 2 - 4 : 0 . 5 mm ratp , lanes 5 - 7 : 0 . 5 mm rutp , lanes 8 - 10 : 0 . 5 mm rgtp , lanes 11 - 13 : 0 . 5 mm ctp and lanes 14 - 16 : 0 . 5 mm datp . in fig2 it is shown that papγ also functions in the aauaaa and cpsf dependent assay . this data provides conclusive proof that poly ( a ) polymerase activity is associated with papγ . the reaction mixture contains in final concentrations : 100 mm tris / hcl buffer ph 8 . 3 ( rt ), 40 mm kcl , 40 μm edta , 9 . 6 % glycerol , 0 . 24 mm dtt , 0 . 9 units rnasin ( ribonuclease inhibitor ), 0 . 01 % np - 40 , 0 . 72 mm mgcl2 , 0 . 2 mg / ml bsa , 1 mm atp , 2 . 5 % pva , 20 mm creatinine phosphate and a mixture radiolabeled rna substrate l3 ( 53 ) ( 70 fmoles in total ). cpsf ( partially purified from calf thymus ) 3 μl , and pap 50 ng are added to the reaction . the reactions proceed at 15 - 30 min ., 30 ° c . the reaction is stopped by proteinase k buffer , the rna is extracted by phenol : chloroformand and it is run in a 10 % acrylamide : bis - acrylamide ( 19 : 1 )- 7 m urea gel . the gel is exposed overnight to a phosphoimager screen and further analyzed . the lanes represent the following : lane 1 : negative control ( no pap ), lane 2 : pap + cpsf . the development of specific polyclonal sera against the unique c - terminal part of the new enabled detection of papγ in hela nuclear extracts ( fig3 ). the proteins from the sds gel are transferred to immobilonp membranes and immunostaining and visualization is done by eclplus reagents . the lanes represent the following : lane 1 : 20 : 14 monoclonal antibody ( 1 : 10 dilution ) against the known human pap gene : three isoforms with apparent mws 90 , 100 , 106 kda . this monoclonal antibody is directed against an epitope with the region of papγ that is shared between paps originating from the two different pap genes . lane 2 , 3 polyclonal sera ( dilution 1 : 2000 , 1 : 4000 respectively ) raised against a polypeptide comprising the c - terminal part located in the unique region starting at approximately amino acid 505 of papγ for specific recognition of one isoform , 90 kda . examples of such unique polypeptide is a polypeptide comprising amino acids located from amino acid 521 to the c - terminal end of papγ . this novel antibody is specific for papγ . this novel antibody was developed against the unique c - terminal part - of papγ . below a detailed description of its production can be found . lane 4 : pre - immune serum ( 1 : 2000 dilution )— no detected signal . cloning of papγ in different cloning vectors for expression in prokaryotic systems the coding sequence of papγ was amplified by pcr using cdna library derived from hela total rna by reverse transcription . the primers used to amplify the first 1479 nt from 5 ′ part were the following : primer ( a ) ( 5 ′- caccatggaagagatgtctgcaaacacc - 3 ′) introducing a ncoi site ( in italics ) upstream of the initiation codon and primer ( d ) ( 5 ′- gagagctcttaggtaccgtgaagttgttttttctttacatgagttgc introducing a saci site downstream of the stop codon ; ( for cloning reasons to the pcal - c vector that will be described further on , we had to introduce a kpni restriction site simultaneously , which introduces 2 extra aa at the c - terminus in all the pet - 32a clones expressing papγ . the ncoi cloning site introduces a point mutation at the second aa in the sequence by changing lys to glutamate )). the pcr product was cloned into pgem - t vector and then by digestion ncoi / saci and ligation was inserted to the pet - 32a ( ncoi / saci digested ); the clone is named pet - 32 ( h1 - 493 )—( where h denotes that the tag is n - terminally located and the numbers 1 - 493 refer to a polypeptide segment starting at amino acid 1 and ending at amino acid 493 of the human pap sequence ). another pair of primers were used to amplify by pcr a fragment reaching up to 2208 nt 3 ′ of papγ ; internal primer ( c ) 5 ′- gcctgtctgggatcctcgggt - 3 ′ and primer ( g ) ( 5 ′- gagagctctaaggtaccttttctttttctttcttcagcagtgcg - 3 ′). pcr product was cloned in pgem - t , digested by ecori / saci and inserted between the ecori and saci restriction enzyme sites of plasmid ppapγ ( h1 - 493 ); the resulting clone is named ppapγ ( h1 - 683 ). for determination of the full length of papγ sequence 3 ′ race methodology was performed according to “ clontech smart race cdna amplification kit ” using two upstream specific primers : primer ( h ) ( caacacctcacaaccctgccca ) and primer ( i ) ( gagatcccattccccatccatag ). the pcr product after seminested was cloned into pgem - t vector and sequenced for confirmation of the identification of stoping codon . the new full - length 3 ′ end of papγ was cloned by a new round of pcr amplification using the pgem - t vector insert and the primer pairs ( c ) and primer ( j ) ( 5 ′- gagaggtaccaagccgattaagggtcagtcg ). the new 3 ′ end was cloned into the same starting clone ppapγ ( h1 - 493 ) by digestion ecori / saci . the pet - 32 ( a ) vector introduces n - terminally of the gene of papγ a thyrodoxine tag which increases the expression of the soluble fraction of the recombinant protein , a histidine tag and an s - tag for enabling easy purification via affinity chromatography . the tag is about 20 kda . the same cloning strategy was used but in the restriction cut the pair ecori / kpni was used . the pcal - c vector introduces a 4 kd calmodulin peptide tag which can be used for affinity purification with calmodulin - affinity resin and results in clones named ppapγ ( 1 - 736c ) or similarly where the numbers indicate encoded papγ amino acids while the c after the numbers denote a c - terminally located tag . expression and purification of recombinant form of pabγ cloned in pet32a vector , in e . coli . papγ containing plasmids were used to transform bl21 ( de3 ) plyss e . coli strains . 1 colony is inoculated in 100 ml tb medium ( containing phosphate ) in the presence of 50 ug / ml carbenicillin and 34 μg / ml chloramphenicol and let grow by standing at 37 ° c . the 100 ml culture is inoculated in a final 1 lt culture in tb medium containing the required antibiotics . bacteria are growing by vigorous shaking at 37 ° c . and are induced at od 600 around 0 . 5 - 1 . 0 with 0 . 42 mm iptg plus 0 . 524 mm mgcl 2 . cells where harvested by centrifugation 3 hours post inducion and pellets frozen at − 70 ° c . extracts were made by thawing the cells on ice and lysing in 50 ml lysis buffer ( 20 mm hepes / koh ph7 . 5 , 0 . 5m kcl , 1 . 0 % np - 40 , 1 . 0 % tween - 20 , 10 % glycerol , 5 mm imidazole , 20 mm β - mercaptoethanol plus 1 tablet of edta - free protease inhibitors . ); next follows sonication ( 3 × 10 sec ), centrifugation 20 min at 39000 g and 0 . 45 μm filtration . the cell extracts are mixed batchwise to 1 ml talon resin ( co ++ affinity agarose ) equilibrated in lysis buffer and proteins bound by 1 hr rotation at 4 ° c . the resin is packed in a manual chromatographic column and washed with 20 column volumes of lysis buffer ; subsequently it is washed with 20 volumes wash buffer ( lysis buffer without detergents and β - mercaptoethanol ). the proteins are eluted by 5 volumes of elution buffer ( 20 mm hepes / koh ph7 . 5 , 0 . 5m kcl , 10 % glycerol , 200 mm imidazole , 0 . 5m kcl ). the eluate is loaded in 1 ml hitrap chelating column equilibrated in 20 mm hepes / koh ph7 . 5 , 0 . 5m kcl , 1 . 0 % np - 40 , 1 . 0 % tween - 20 , 10 % glycerol , 50 mm imidazole ). the column is washed with 10 column volumes equilibration buffer and 10 volumes wash buffer ( equilibration buffer without detergents and containing 0 . 05 m kcl ). the proteins are eluted with 5 volumes elution buffer ( 20 mm hepes / koh ph 7 . 5 , 0 . 05m kcl , 10 % glycerol , 0 . 5 mm dtt , 1 . 5 mm mgcl2 , 200 mm imidazole ). the eluate is loaded to a heparin hi - trap column equilibrated in 20 mm hepes / koh ph 8 . 6 , 0 . 05m kcl , 10 % glycerol , 0 . 5 mm dtt , 1 . 5 mm mgcl2 buffer . it is washed with 5 volumes of the equilibration buffer and recombinant proteins eluted in fractions of 0 . 5 ml with 5 volumes of the same buffer containing 0 . 5 m kcl . in all buffer solutions they are added freshly 0 . 5 mm pmsf , 1 . 0 μg / ml leupeptin , 1 . 0 mg / ml pepstatin , 1 . 0 μg / ml aprotinin . expression and purification of recombinant form of pabγ cloned in pcal - c vector , in e . coli . pcal - c papγ containing plasmids were used to transform bl21 ( de3 ) plyss e . coli strains . 1 colony is inoculated in 50 ml tb medium ( containing phosphate buffer ) plus 50 μg / ml carbenicillin and let grow by standing at 37 ° c . the 50 ml culture is inoculated in a final 500 ml culture in tb medium containing antibiotics . bacteria are growing by vigorous shaking at 37 ° c . and induced at od 600 around 0 . 6 - 1 . 0 with 0 . 42 mm iptg plus 0 . 524 mm mgcl 2 . cells where harvested by centrifugation 3 hours post induction and pellets frozen at − 70 ° c . extracts where made by unthawing the cells on ice and lysing in 30 ml lysis buffer —( ca - binding buffer ) ( 50 mm tris / hcl ph7 . 5 , 0 . 15 m kcl , 0 . 1 % triton x - 100 , 10 % glycerol , 1 mm mg ( ch 3 coo ), 2 mm cacl 2 , 1 mm imidazole , 10 mm β - mercaptoethanol plus 1 tablet of edta - free protease inhibitors . ); next follows sonication ( 4 × 30 sec ), centrifugation 20 min at 39000 g and 0 . 45 μm filtration . the cell extracts are mixed batchwise to 0 . 75 ml calmodulin resin ( affinity agarose ) equilibrated in binding buffer and proteins bound by rotation at 4 ° c . overnight . the resin is packed in a manual chromatographic column and washed with 20 column volumes of wash buffer i ( 50 mm tris / hcl ph7 . 5 , 0 . 2 m kcl , 0 . 1 % triton x - 100 , 10 % glycerol , 1 mm mg ( checoo ), 2 mm cacl 2 , 1 mm imidazole , 10 mm β - mercaptoethanol ; subsequently it is washed with 20 volumes washii buffer ( 50 mm tris / hcl ph7 . 5 , 0 . 25 m kcl , 10 % glycerol , 1 mm . mg ( ch 3 coo ), 2 mm cacl 2 , 1 mm imidazole ). the recombinant protein is eluted in fractions of 0 . 5 ml with 7 volumes of buffer containing 50 mm tris / hcl ph 7 . 5 , 1 m kcl , 2 mm egta , 10 % glycerol , 0 . 5 mm dtt and 1 . 5 mm mgcl 2 . in all buffer solutions they are added freshly 0 . 5 mm pmsf , 1 . 0 μg / ml leupeptin , 1 . 0 mg / ml pepstatin , 1 . 0 μg / ml aprotinin . a unique part of the papγ sequence , as represented by a polypeptide starting at amino acid 521 and ending at amino acid 683 , was cloned in pet - 32 ( a ) vector and the recombinant polypeptide was purified and used for immunization of rabbits . the 491 nt long fragment , comprising the above mentioned amino acids , was amplified using as template the plasmid pet - 32 ( 668 ) and a pair of primers : primer ( p ) ( 5 ′- caccatggaatccaaaagattgtctctggatagc - 3 ′) and primer ( g ) ( 5 ′- gagagctcttaggtaccttattttctttttctttcttcagcagtgcg - 3 ′). the pcr product is cloned to pgem - t vector and inserted to pet32 ( a ) vector after restriction digestion with ncoi / bamhi . the recombinant polypeptide is expressed and purified , as described essentially , at the recombinant proteins &# 39 ; purification schedule . the antigen was more than 95 % pure , was diluted to 0 . 9 mg / ml protein concentration . 500 μl of antigen were used for injection of 2 independent rabbits in repetitive injections . ballantyne , s ., bilger a ., { dot over ( a )} ström , j ., virtanen , a . and wickens , m . 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( 1996 ) complex alternative rna processing generates an unexpected diversity of poly ( a ) polymerase isoforms . mol . cell . biol ., 16 , 2378 - 2386w . met lys glu met ser ala asn thr val leu asp ser gln arg gln gln lys his tyr gly ile thr ser pro ile ser leu ala ser pro lys glu ile asp his ile tyr thr gln lys leu ile asp ala met lys pro phe phe thr phe gly ser tyr arg leu gly val his thr lys gly ala asp ile asp ala leu cys val ala pro arg his val glu arg ser asp phe phe gln ser phe phe glu lys leu lys his gln asp gly ile arg asn asp gly ile glu ile asp leu val phe ala arg leu ala ile gln thr asp ile arg cys ile arg ser leu asn gly cys arg val thr asp glu ile leu his leu val pro asn lys glu thr phe arg leu thr leu arg ala val lys leu trp ala lys arg arg gly ile tyr ser asn met leu gly phe leu gly gly val ser trp ala met leu val ala arg thr cys gln leu tyr pro asn ala ala ala ser thr leu val his lys phe phe pro ser asp arg tyr his leu met pro ile ile thr pro ala tyr pro val glu glu phe lys gln gly leu ala val thr asp glu ile leu gln gly lys ser asp trp ser lys leu leu glu pro pro asn phe phe gln gly asn leu glu arg asn glu phe ile thr leu ala his val asn pro gln ser phe pro gly asn lys glu his his lys asp asn asn tyr val ser met trp phe leu gly ile ile phe arg arg val glu asn ala glu val tyr arg gln ala asn asn ile asn met leu lys glu gly met lys ile glu ala thr his val lys lys lys gln leu his his tyr leu pro pro ala gln gly gln pro his leu asn gly met ser asn ile thr lys met lys glu met ser ala asn thr val leu asp ser gln arg gln gln lys his tyr gly ile thr ser pro ile ser leu ala ser pro lys glu ile asp his ile tyr thr gln lys leu ile asp ala met lys pro phe phe thr phe gly ser tyr arg leu gly val his thr lys gly ala asp ile asp ala leu cys val ala pro arg his val glu arg ser asp phe phe gln ser phe phe glu lys leu lys his gln asp gly ile arg asn asp gly ile glu ile asp leu val phe ala arg leu ala ile gln thr asp ile arg cys ile arg ser leu asn gly cys arg val thr asp glu ile leu his leu val pro asn lys glu thr phe arg leu thr leu arg ala val lys leu trp ala lys arg arg gly ile tyr ser asn met leu gly phe leu gly gly val ser trp ala met leu val ala arg thr cys gln leu tyr pro asn ala ala ala ser thr leu val his lys phe phe pro ser asp arg tyr his leu met pro ile ile thr pro ala tyr pro val glu glu phe lys gln gly leu ala val thr asp glu ile leu gln gly lys ser asp trp ser lys leu leu glu pro pro asn phe phe gln lys tyr arg his tyr ile val leu thr ala ser ala ser thr glu glu asn his leu glu trp val gly leu val glu ser lys ile arg val leu val gly asn leu glu arg asn glu phe ile thr leu ala his val asn pro gln ser phe pro gly asn lys glu his his lys asp asn asn tyr val ser met trp phe leu gly ile ile phe arg arg val glu asn ala glu ser val asn ile asp leu thr tyr asp ile gln ser phe thr asp thr val tyr arg gln ala asn asn ile asn met leu lys glu gly met ala gln gly gln pro his leu asn gly met ser asn ile thr lys thr