Patent Application: US-95891402-A

Abstract:
biologically active molecules are produced from their biologically inactive precursors by mutation of their native cleavage site to a site which is capable of being cleaved by a protease , and cleavage of the mutated molecule to yield a biologically active molecule .

Description:
according to the present invention , a cdna encoding human proil - 18 in which the caspase - 1 cleavage site was mutated into a factor xa site , was prepared . other mutations , generating cleavage sites suitable for other proteases may be used . leu - val - pro - arg - gly - ser ( seq id no : 1 ), a sequence cleaved between the amino acids arg and gly by the protease thrombin . ile - glu - gly - arg -( seq id no : 2 ), a sequence cleaved after the arg residue by the protease factor xa . asp - asp - asp - lys -( seq id no : 3 ), a sequence cleaved after the lys residue by the protease enterokinase . his - tyr -( seq id no : 4 ), a sequence cleaved after the tyr residue by the protease subtilisin or by its variant genenase ™ ( new england biolabs ). tyr - his ( seq id no : 5 ), a sequence cleaved after the tyr residue by the protease subtilisin or by its variant genenase ™ ( new england biolabs ). leu - glu - val - leu - phe - gln - gly - pro ( seq id no : 6 ), a sequence cleaved after the gln residue by the human rhinovirus 3c protease . similarly , a cdna encoding human proil - 1β in which the caspase - 1 cleavage site is mutated into a factor xa site is prepared . other mutations , generating cleavage sites suitable for other proteases may be used . ile - glu - gly - arg -( seq id no : 2 ), a sequence cleaved after the arg residue by the protease factor xa . asp - asp - asp - lys -( seq id no : 3 ), a sequence cleaved after the lys residue by the protease enterokinase . leu - glu - val - leu - phe - gln - gly - pro ( seq id no : 6 ), a sequence cleaved after the gln residue by the human rhinovirus 3c protease . the choice of mutation depends on the following parameters : the cleavage site generated by the mutation must be very specific to avoid undesired cleavages at other sites within the polypeptide backbone of the proil - 18 or proil - 1β ; the protease must exhibit a high level of specificity for the engineered cleavage site ; the protease must be conveniently available , e . g ., from commercial sources ; and the mutation preferably should not introduce a major alteration in the secondary or tertiary structure of the proil - 18 or proil - 1β . the use of factor xa or enterokinase or subtilisin is preferable over the other proteases because the resulting il - 18 or il - 1β has no mutated amino acid . the use of factor xa is most favorable because it requires the most conservative mutations , thereby reducing the risk of altered secondary or tertiary structure of the proil - 18 or proil - 1β and the risk that cleavage will not take place . the use of factor xa or enterokinase is preferable over the other proteases because the resulting il - 18 or il - 1β have no mutated amino acids . in order to construct a cdna encoding a human proil - 18 mutant ( proil - 18 iegr ) in which the caspase - 1 cleavage site is mutated into a factor xa cleavage site , the following mutations were introduced in the human proil - 18 cdna : l33i ; s35g and d36r . these mutations yielded the factor xa recognition sequence iegr . a vector expressing human proil - 18 iegr was constructed in the following manner . peripheral blood mononuclear cells ( pbmc ) of a healthy donor were stimulated by bacterial lipopolysaccharide ( lps ). total rna was extracted from the cells . the rna was reverse - transcribed and the resulting cdna served as a template for polymerase chain reaction ( pcr ). the mutation in the sequence was introduced using overlapping primers in three steps of pcr ( fig1 ). the resulting 600 bp pcr product , coding for mutated human proil - 18 was cloned into a ta cloning vector ( pgem - t easy , promega ) and its dna sequence was verified by dna sequence analysis . the resulting pgem - t - proil - 18 iegr plasmid was cut - by ecor i and bamh i and the 600 bp fragment encoding human proil - 18 iegr was subcloned into the prokaryotic expression vector pgex - 2tk containing the gst gene sequence ( pharmacia ). the resulting expression vector contained the sequence of the human proil - 18 iegr gene fused in frame to the 3 ′ end of the gst gene ( fig2 ). expression and purification of gst - proil - 18 iegr was then performed . a fresh single colony of e . coli jm109 transformed with plasmid pgex - proil - 18 iegr was grown with shaking at 37 ° c ., until reaching a density of 0 . 6 od 600 . protein expression was then induced by isopropyl - thiogalactoside ( iptg ) and further culturing at 37 ° c . for 3 h . sds - page of total bacterial proteins revealed the induction of a 43 kd protein , corresponding in size to gst - proil - 18 iegr ( fig3 ). all downstream steps for isolation of the gst - proil - 18 iegr were carried out at 4 ° c . bacteria were harvested by centrifugation , resuspended in phosphate buffered saline ( pbs ), containing protease inhibitors . the bacteria were lysed by sonication , triton x - 100 was added to a final concentration of 1 % and the clarified lysate was mixed with glutathione - agarose beads ( pharmacia ). the beads were washed and an aliquot of the beads was analyzed by sds - page . a single 43 kd band , corresponding to the gst - proil - 18 iegr fusion protein was found ( fig4 ). the beads were then digested with factor xa and the supernatant was collected . sds - page of the supernatant gave a major band of approximately 18 kd , in agreement with the expected mass of mature human il - 18 . an additional weaker band of 19 . 5 kd was seen as well ( fig5 ). protein sequence analysis of the 18 kd and the 19 . 5 kd bands gave in both cases the expected n - terminal sequence of mature human il - 18 . a 43 kd band , corresponding to the gst - proil - 18 iegr fusion protein was still present in the glutathione - agarose beads following digestion , indicating an incomplete cleavage ( not shown ). the recombinant human il - 18 was assayed for biological activity in murine splenocytes , based on its ability to induce the expression of interferon - gamma . incubation of mouse non - adherent spleen cells with human il - 18 alone showed little ifn - gamma production , suggesting a requirement for a co - stimulant . con a alone at 0 . 125 μg / ml failed to induce significant levels of ifn - γ . when the non - adherent splenocytes were incubated with con a and il - 18 of the present invention , a dose - dependent induction of ifn - γ was obtained ( fig6 ). in order to construct a cdna encoding a human proil - 1β mutant ( proil - 1β iegr ) in which the caspase - 1 cleavage site is mutated into a factor xa cleavage site , the following mutations are introduced in the human proil - 1β cdna : y1131 , v114e , h115g and d116r . these mutations yield the factor xa recognition sequence iegr . the following steps are taken in order to construct a vector expressing human proil - 1β iegr . peripheral blood mononuclear cells ( pbmc ) of a healthy donor are stimulated by bacterial lipopolysaccharide ( lps ). total rna is extracted from the cells . the rna is reverse - transcribed and the resulting cdna serves as a template for polymerase chain reaction ( pcr ) with primers corresponding to the coding sequence of human proil - 1β . the resulting pcr product is cloned into a ta cloning vector , e . g ., pgem - t easy from promega , and then subjected to site - directed mutagenesis with a suitable oligonucleotide . the sequence of the resulting vector , coding for mutated human proil - 1β , is verified by sequence analysis . the resulting pgem - t - proil - 1β iegr plasmid is cut by proper restriction enzymes and the ˜ 820 bp fragment encoding human proil - 1β iegr is subcloned into the prokaryotic expression vector pgex - 2tk ( pharmacia ). the resulting expression vector contains the sequence of the human proil - 1β iegr gene fused in frame to the 3 ′ end of the gst gene . expression and purification of gst - proil - 1β iegr is similarly performed . a fresh single colony of e . coli jm109 transformed with plasmid pgex - proil - 1β iegr is grown with shaking at 37 ° c ., until reaching a density of 0 . 6 od 600 . protein expression is then induced by isopropyl - thiogalactoside ( iptg ) and further culturing at 37 ° c . for 3 h . sds - page of total bacterial proteins reveals the induction of a ˜ 52 kd protein , corresponding in size to gst - proil - 1β iegr . all downstream steps for isolation of the gst - proil - 1β iegr are carried out at 4 ° c . bacteria are harvested by centrifugation , resuspended in phosphate buffered saline ( pbs ), containing protease inhibitors . the bacteria are lysed by sonication , triton x - 100 is added to a final concentration of 1 % and the clarified lysate is mixed with glutathione - agarose beads ( pharmacia ). the beads are washed and an aliquot of the beads is analyzed by sds - page for the presence of the ˜ 52 kd gst - il - 1β fusion protein . the beads are then digested with factor xa and the supernatant containing mature human il - 1β is collected . in another embodiment of the present invention , the expression and purification of a polypeptide is obtained by producing a fusion protein having the cleavage site for a caspase . a plasmid expressing a binding peptide fused to a protein of interest can be mutated so that a caspase cleavage site is introduced in a position convenient for the purification of an active polypeptide . caspase recognition sites are composed of 4 amino acids , the last one being an aspartic acid . generally , caspases show an higher and different specificity for the cleavage sites than commonly used proteases , offering significant advantages towards proteases previously described as useful for cutting fusion peptides . the purification procedure is examplified in fig7 . preferred cleavage sites for each caspase are described in literature ( ann rev . biochem , 1999 , 68 : 383 – 424 ). all of them have the residue asp in common in the pi position . the plasmid described above , pgex - proil - 18 iegr , was modified to obtain a different human proil - 18 mutant ( proil - 18 letd , see fig8 ) in which the factor xa cleavage site is mutated into a caspase - 8 cleavage site . this allows the use of caspase - 8 for cleavage . another advantage of using a caspase is their high efficiency , in terms of digestion yield and digestion time . it was found that when comparing the il - 18 protein obtained from pgex - proil - 181egr and pgex - proil - 18letd , the amount of enzyme and the time needed to obtain 1 mg of hil - 18 is reduced . the procedure lead to the production of a protein of the expected size ( fig9 ) and bioactivity . caspase - 8 is not the only suitable caspase and , according to the presence or absence of a specific caspase cleavage site in a protein of interest to purify or any other criteria , the mutations corresponding to the cleavage site of any other caspase can be used . the caspase can be removed from the protein of interest using standard purification methods , such as e . g . gel filtration or ion - exchange chromatography . in conclusion , the method described here provides purified , biologically active human proteins , such as il - 18 and il - 1β from e . coli . it appears that correct folding of these cytokines requires expression as a precursor first , and then cleavage to its mature form . human il - 18 differs in this respect from murine il - 18 , which may be expressed in host cells as a mature , biologically active cytokine . the invention will now be illustrated by the following non - limiting examples : construction of a plasmid coding for human pro il - 18 with a factor xa site pbmc were obtained from peripheral blood of a healthy volunteer by ficoll - paque plus ( pharmacia ) density gradient separation . the pbmc were washed twice with ice - cold pbs containing 2 % fbs and resuspended at 2 . 5 × 10 6 cells / ml in rpmi - 1640 medium , supplemented with fetal bovine serum ( fbs ), 2 mm l - glutamine , 100 u / ml penicillin and 100 u / ml streptomycin ( rpmi - 10 ). the culture was stimulated with lps ( 1 μg / ml , 3 h , 37 ° c .). total rna was extracted from the cells with the tripure ™ isolation reagent ( boehringer mannheim ). one μg total rna was used for rt - pcr with the titan one tube rt - pcr kit ( boehringer mannheim ) according to the manufacturer &# 39 ; s instructions . proil - 18 iegr cdna was constructed by a three - step pcr , using the overlapping primer method . the various primers were designed on the basis of the human proil - 18 cdna ( genbank accession no . d49950 , ( 3 )). 5 ′- aacatcgaaggtcgttactttggcaagcttgaatc - 3 ′, ( seq id no : 7 ) and the reverse primer was 5 ′- cgcgaattcctagtcttcgttttgaacagt - 3 ′ ( seq id no : 8 ). an eco ri site was included in the reverse primer . the template was a reverse - transcribed total cellular rna , isolated from the lps - stimulated pbmc . the thermocycle conditions were : 10 cycles of 94 ° c ., 30 sec ; 55 ° c ., 30 sec and 68 ° c ., 2 min followed by 25 cycles of 94 ° c ., 30 sec ; 55 ° c ., 30 sec and 68 ° c ., 125 sec / cycle . the resulting 498 bp pcr product was amplified in the second pcr step using the overlapping forward primer : 5 ′- tggcaatgaaatttattgacaatacgctttactttatagctgaagatgatga aaacatcgaaggtcgttactttg - 3 ′ ( seq id no : 9 ), and the reverse primer of the previous step . the thermocycling conditions were : 30 cycles of 94 ° c ., 30 sec ; 56 ° c ., 30 sec and 72 ° c ., 1 min . the resulting 551 bp product was amplified in the third step using the overlapping forward primer : 5 ′- cgtggatccatggctgctgaaccagtagaagacaattgcatcaactttgtgg caatgaaatttattgac - 3 ′ ( seq id no : 10 ) and the reverse primer of the previous steps . a bamh i site was included in the forward primer . the thermocycling conditions were the same as those of the previous step . the resulting 0 . 6 kb pcr product ( fig1 ) was purified by a high pure ™ pcr product purification kit ( boehringer mannheim ) and ligated into the pgem - t easy vector to yield plasmid pgem - proil - 18 iegr . ligation products were transformed into jm109 competent cells ( promega corporation ). standard cloning protocols were used throughout ( 11 ). plasmid pgem - proil - 18 iegr from a single colony was verified for the presence of a correct insert by restriction enzyme digestion and dna sequencing . the plasmid was digested with bamh i and ecor i . the resulting bamh i / ecor i dna encoding human proil - 18 iegr was resolved by electrophoresis in 1 . 0 % agarose and the 0 . 6 kb band was isolated from the gel by a qiaquick gel extraction kit ( diagen , germany ). this dna was ligated into the pgex - 2tk expression vector ( pharmacia ) which was linearized with bamh i and ecor i . the resulting recombinant plasmid pgex - proil - 18 iegr ( fig2 ) was transformed into e . coli jm109 strain and the resulting vector was confirmed by restriction enzyme digestion and nucleic acid sequencing . a 50 ml overnight culture from a fresh single colony of e . coli jm109 transformed with plasmid pgex - proil - 18 iegr was diluted in 450 ml lb medium containing 100 u / ml ampicillin and grown with shaking at 37 ° c ., until reaching a density of 0 . 6 od 600 . protein expression was then induced by adding iptg to a final concentration of 0 . 1 mm and further culturing at 37 ° c . for 3 h . bacteria were harvested by centrifugation ( 5 , 000 × g , 5 min , 4 ° c .) and rapidly resuspended in a 1 : 100 starting volume of ice - cold phosphate buffered saline ( pbs ; 150 mm nacl , 16 mm na2hpo4 , 4 mm nah2po4 , ph 7 . 3 ), containing the protease inhibitors ( leupeptin 1 μg / ml , pepstatin a 1 μg / ml , aprotinin 2 μg / ml , pmsf 0 . 2 mm and edta 5 mm ). cells were lysed on ice by mild sonication ( 4 × 15 sec bursts ). triton x - 100 was added to a final concentration of 1 % and the mixture kept on ice for 5 min . the clarified lysate ( 14 , 000 × g , 15 min , 4 ° c .) was mixed with a 50 % suspension of glutathione - agarose beads ( 2 ml , pharmacia ) and the mixture was incubated with mild shaking at 4 ° c . for 3 h . the beads were pelleted by centrifugation ( 500 × g , 5 min ), washed twice with 10 volumes of lysis buffer ( 1 % triton x - 100 in pbs ), twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , 150 mm nacl , and twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , 150 mm nacl , 2 . 0 mm cacl2 ( cleavage buffer ). all steps were carried out at 4 ° c . the glutathione - agarose beads with bound gst - proil - 18 iegr fusion protein were incubated ( 6 h , 23 ° c .) with factor xa ( new england biolabs ) in 1 ml cleavage buffer , using a protease to substrate ratio of 1 : 50 ( w / w ), with constant mixing on a rotating wheel . this step releases the mature human il - 18 . the beads were pelleted by centrifugation at 500 × g for 5 minutes at 4 ° c . and washed 5 times with 2 ml ice - cold pbs . the supernatant and all the washing volumes were combined , clarified by centrifugation ( 14 , 000 × g , 15 min ., 4 ° c .) and concentrated by ultrafiltration ( centricon - 10 , amicon ). aliquots of the protein , as well as beads before and after digestion were resolved by 0 . 1 % sds - 12 % polyacrylamide gel electrophoresis followed by coomassie blue staining . a major band of approximately 18 kd , in agreement with the expected mass of mature human il - 18 , was obtained in the supernatant . a minor band of 19 . 5 kd was seen as well ( fig5 ). n - terminal protein sequence analysis of the two bands gave the sequence yfgk . . . , in accordance with that of mature human il - 18 . the purified protein was sterilized through a 0 . 2 μ filter and stored at − 70 ° c . human il - 18 activity was assessed as described ( 2 ). briefly , spleens of normal c3h / hej mice were excised , minced and exposed to gey buffer ( 0 . 144 m nh4cl in 0 . 017 m tris - hcl ph 7 . 2 ) to disrupt erythrocytes . the white cells were washed twice with rpmi - 5 and then resuspended in rpmi - 10 . for the preparation of plastic - non - adherent cells , cell suspensions were incubated ( 60 min ., 37 ° c .) in 100 mm plastic plates ( becton dickinson ltd ., uk ). non - adherent cells were gently gathered , centrifuged and resuspended in rpmi - 10 . viable cells were counted by the trypan blue dye exclusion test and the cell concentration was adjusted to 5 × 10 6 cells / ml . most of the non - specific esterase - positive cells were removed by these procedures . suspensions of plastic - non - adherent cells ( 0 . 15 ml ) were placed in 96 - well flat - bottom microtiter culture plate ( nunclon ™, denmark ). various concentrations of il - 18 , polymyxin ( 1μg / ml ) and con a ( 0 . 125 μg / ml ) were added to the wells in 50 μl rpmi - 10 . the plates were incubated for 24 or 48 h at 37 ° c . in 5 % co2 . following incubation , supernatants were collected and murine ifn - γ titers were determined by elisa . incubation of mouse non - adherent spleen cells with human il - 18 alone showed little ifn - γ production , suggesting a requirement for a co - stimulant . con a alone at 0 . 125 μg / ml failed to induce significant levels of ifn - γ . when the non - adherent splenocytes were incubated with con a and the human il - 18 of the present invention , a dose - dependent induction of ifn - γ was obtained ( fig6 ). construction of a plasmid coding for human pro il - 1β with a factor xa site pbmc are obtained from peripheral blood of a healthy volunteer by ficoll - paque plus ( pharmacia ) density gradient separation . the pbmc are washed twice with ice - cold pbs containing 2 % fbs and resuspended at 2 . 5 × 10 6 cells / ml in rpmi - 1640 medium , supplemented with fetal bovine serum ( fbs ), 2 mm l - glutamine , 100 u / ml penicillin and 100 u / ml streptomycin ( rpmi - 10 ). the culture is stimulated with lps ( 1 μg / ml , 3 h , 37 ° c .). total rna is extracted from the cells with the tripure ™ isolation reagent ( boehringer mannheim ). one μg total rna is used for rt - pcr with the titan one tube rt - pcr kit ( boehringer mannheim ) according to the manufacturer &# 39 ; s procedure . proil - 1β cdna is cloned by pcr with sense and reverse primers designed on the basis of the human proil - 1β cdna ( genbank accession no . m15330 ). the sense primer corresponds to nucleotides 87 – 107 of the sequence m15330 , preceded by a bamh i cleavage site . the reverse primer corresponds to nucleotides 896 – 876 of the sequence m15330 , preceeded by an eco ri site . the template is a reverse - transcribed total cellular rna , isolated from the lps - stimulated pbmc . the thermocycle conditions are : 10 cycles of 94c , 30 sec ; 55 ° c ., 30 sec and 68 ° c ., 2 min followed by 25 cycles of 94 ° c ., 30 sec ; 55 ° c ., 30 sec and 68 ° c ., 125 sec / cycle . the resulting 830 bp pcr product is purified by a high pure ™ pcr product purification kit ( boehringer mannheim ) and ligated into the pgem - t easy vector to yield plasmid pgem - proil - 1β . the plasmid is subjected to site - directed mutagenesis with the oligonucleotide acatgggataacgaggctattgaaggccgggcacctgtacga ( seq id no : 11 ), corresponding to nucleotides 405 – 452 of human il - 1β mrna and having the mutations y1131 , v114e , h115g and d116r . the resulting plasmid pgem - proil - 1β iegr from a single colony is verified for the presence of a correct insert by restriction enzyme digestion and dna sequencing . the plasmid is digested with bamh i and ecor i . the resulting bamh i / ecor i dna encoding human proil - 1β iegr is resolved by electrophoresis in 1 . 0 % agarose and the 0 . 82 kb band is isolated from the gel by a qiaquick gel extraction kit ( diagen , germany ). this dna is ligated into the pgex - 2tk expression vector ( pharmacia ) which is linearized with bamh i and ecor i . the resulting recombinant plasmid pgex - proil - 1β iegr is transformed into e . coli jm109 strain and the resulting vector is confirmed by restriction enzyme digestion and nucleic acid sequencing . a 50 ml overnight culture from a fresh single colony of e . coli jm109 transformed with plasmid pgex - proil - 1β iegr is diluted in 450 ml lb medium containing 100 u / ml ampicillin and grown with shaking at 37 ° c ., until reaching a density of 0 . 6 od 600 . protein expression is then induced by adding iptg to a final concentration of 0 . 1 mm and further culturing at 37 ° c . for 3 h . bacteria are harvested by centrifugation ( 5 , 000 × g , 5 min , 4 ° c .) and rapidly resuspended in a 1 : 100 starting volume of ice - cold phosphate buffered saline ( pbs ; 150 mm nacl , 16 mm na2hpo4 , 4 mm nah2po 4 , ph 7 . 3 ), containing the protease inhibitors ( leupeptin 1 μg / ml , pepstatin a 1 μg / ml , aprotinin 2 μg / ml , pmsf 0 . 2 mm and edta 5 mm ). cells are lysed on ice by mild sonication ( 4 × 15 sec bursts ). triton x - 100 is added to a final concentration of 1 % and the mixture kept on ice for 5 min . the clarified lysate ( 14 , 000 × g , 15 min , 4 ° c .) is mixed with a 50 % suspension of glutathione - agarose beads ( 2 ml . pharmacia ) and the mixture is incubated with mild shaking at 4 ° c . for 3 h . the beads are pelleted by centrifugation ( 500 × g , 5 min ), washed twice with 10 volumes of lysis buffer ( 1 % triton x - 100 in pbs ), twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , 150 mm nacl , and twice with 10 volumes of 20 mm tris - hcl , ph 8 . 0 , 150 mm nacl , 2 . 0 mm cacl2 ( cleavage buffer ). all steps are carried out at 4 ° c . the glutathione - agarose beads with bound gst - proil - 1β iegr fusion protein are incubated ( 6 h , 23 ° c .) with factor xa ( new england biolabs ) in 1 ml cleavage buffer , using a protease to substrate ratio of 1 : 50 ( w / w ), with constant mixing on a rotating wheel . this step releases the mature human il - 1β . the beads are pelleted by centrifugation at 500 × g for 5 minutes at 4 ° c . and washed 5 times with 2 ml ice - cold pbs . the supernatant and all the washes are combined , clarified by centrifugation ( 14 , 000 × g , 15 min ., 4 ° c .) and concentrated by ultrafiltration ( centricon - 10 , amicon ). aliquot of the protein , as well as beads before and after digestion are_resolved by 0 . 1 % sds - 12 % polyacrylamide gel electrophoresis followed by coomassie blue staining . a major band of approximately 17 kd , in agreement with the expected mass of mature human il - 1β , is obtained in the supernatant . the purified protein is sterilized through a 0 . 2 μ filter and stored at − 70 ° c . construction of a plasmid coding for human pro il - 18 with a caspase - 8 cleavage site the recombinant plasmid pgex - proil - 18 iegr of example 1 was modified by inserting a pcr amplified dna fragment with the caspase - 8 cleavage site ( letd ). in brief , pgex - proil - 18 iegr was digested with the restriction enzymes bam h1 at pos 951 and hind iii at pos 1074 cutting out the dna portion containing the pro - domain , the factor xa cleavage site ( iegr ) and three amino acids from the n - terminus of hil - 18 . the same plasmid was used as template for the pcr amplification , by introducing the mutated site letd on dowstream primer : atgcaag ctt gcc aaa gta gtc ggt ttc cag gtt ttc atc atc ttc agc tat aa ( seq id no : 12 ). the pcr fragment was purified using the “ high pure product purification ” kit ( boehringer mannheim ) then similarly cut with bam hi and hind iii generating a 123 bp insert . the open vector and the insert were separated on agarose gel and extracted using the commercial kit “ jet sorb ” ( genomed ). ligation was done overnight using a t4 ligase ( new england biolabs , beverly mass ., usa ) and the ligation products were transformed into e . coli dh5α competent cells . several colonies were isolated , subcultured and miniprep prepared with the & lt ;& lt ; qiagen & gt ;& gt ; kit . pcr amplification ( perkin elmer gene amp - amplitaq dna polymerase ) was done on all the minipreps in order to sequence the essential part of the recombinant plasmid . final transformation was done in competent bacteria e . coli jm109 following the tss method ( chung , c . t . et al . ( 1989 ) proc . natl . acad . sci . u . s . a . 86 , 2172 – 2175 ). using the plasmid constructed according to example 8 above , gst - pro - letd - hil18 protein expression was achieved in shake flasks and 5 liter fermenters essentially as described in example 2 above . cleavage of the fusion protein using caspase - 8 and purification of mature human il - 18 the glutathione - agarose beads with bound gst - pro - il - 18 letd fusion protein were incubated ( 4 h , 23 ° c .) with caspase - 8 ( calbiochem ) in cleavage buffer ( defined in example 6 ) using a protease : substrate ratio of 1 : 1200 ( w / w ) with constant mixing on a rotating wheel or roller . the beads were pelleted by centrifugation at 500 × g for 5 minutes at 4 ° c . and washed 5 times with 2 ml ice - cold pbs . the supernatant and all the washing volumes were combined , clarified by centrifugation ( 14 , 000 × g , 15 min ., 4 ° c .) and concentrated by centrifugation ( centricon - 10 , amicon ). the concentrated digest was then submitted to molecular sizing on superdex - 75 ( pharmacia ) in order to separate the hil - 18 ( 18 kda ) from higher molecular weight cleavage products and from the residual proteolytic enzyme ( caspase - 8 heterodimer , 29 kda and hetero - tetramers , 58 kda ). caspase - 8 enzymatic activity was assayed in the various superdex - 75 fractions using the caspase - 8 assay kit and the ac - ietd - amc fluorogenic substrate ( biomol ) and was observed in fractions corresponding to a molecular mass of 55 kd ( corresponding to the caspase - 8 hetero - tetramer ), showing therefore to be very well separated from fractions containing hil - 18 ( 17 kd ). the highly pure 18 kda hil - 18 gel filtration fractions were pooled , concentrated on centricon - 10 ( amincon ) and finally sterilized through a 0 . 22 μm filter and stored at − 80 ° c . aliquots of the protein , as well as the beads before and after digestion were resolved by 0 . 1 % sds - 12 % polyacrylamide gel electrophoresis followed by coomassie blue staining ( fig9 ). a band of 18 kd , in agreement with the expected mass of mature human il - 18 , was observed in the supernatant fraction . analysis of the bead fraction showed that the cleavage is not complete , since precursor protein remains bound to the beads . furthermore , some il - 18 cleaved protein remains in the bead fraction . human il - 18 activity was assessed as described ( 10 ). briefly , kg - 1 cells were maintained in dmem containing 20 % fbs . for the il - 18 assay , kg - 1 cells were suspended at 1 . 2 × 10 6 cells / ml ( 250 μl / well , 48 well plate ) and stimulated with mtnfα ( innogenetics ) together with various concentration of hil - 18 ( serial dilutions , starting from 80 ng / ml ). the plate was incubated 24 h at 37 ° c . in 5 % co 2 . following incubation , supernatants were collected and murine ifn - γ content was determined by elisa ( rec . hifn - γ and anti - hifn - γ from r & amp ; d systems ). human il - 18 induced a dose dependent production of ifn - γ with identical activity to the hil - 18 obtained from the factor xa cleavage site construct . 1 . nakamura , k ., okamura , h ., nagata , k ., komatsu , t ., and tamura , t . 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