Patent Application: US-2367308-A

Abstract:
the present invention relates to a novel human poly polymerase 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 named papγ 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 . 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 ′) ( seq id no : 5 ) introducing a ncoi site ( in italics ) upstream of the initiation codon and primer ( d ) ( 5 ′ gagctcttaggtaccgt gaagttgttttttctttacatgagttgc ( seq id no : 6 ) 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 ′- gcctgtctgggat cctcgggt - 3 ′ ( seq id no : 7 ) and primer ( g )( 5 ′- gagagctctaag gtaccttttctttttctttcttcagcagtgcg - 3 ′) ( seq id no : 8 ). 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 ) ( seq id no : 9 ) and primer ( i ) ( gagatcccattccccatccatag ) ( seq id no : 10 ). the pcr product after seminested was cloned into pgem - t vector and sequenced for confirmation of the identification of stop 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 ) seq id no : 11 ). the new 3 ′ end was cloned into the same starting clone ppapγ ( h1 - 493 ) by digestion with ecori / saci . 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 ′- caccatggaatccaaaa gattgtctctggatagc - 3 ′) ( seq id no : 12 ) and primer ( g )( 5 ′- gagag ctcttaggtaccttattttctttttctttcttcagcagtgcg - 3 ′) ( seq id no : 13 ). 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 ; 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