# Patent

## Title
Processing enzymes fused to basic protein tags

## Abstract

The invention is related to processing enzyme comprising an N-terminally attached tag derived from highly basic proteins from thermophilic bacteria. The processing enzymes are useful for modifying proteins. They can be produced in high yields and can be effectively separated from the modified protein after use.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of U.S. patent application Ser. No. 12/944,400, filed Nov. 11, 2010, which is a divisional application of U.S. patent application Ser. No. 12/443,694, filed Mar. 31, 2009, which is 35 U.S.C. §371 national stage application of International Patent Application PCT/EP2007/060908 (published as WO 2008/043847 A1), filed Oct. 12, 2007, which claimed priority of European Patent Application 06122266.7, filed Oct. 13, 2006; this application further claims priority under 35 U.S.C. §119 of U.S. Provisional Application 60/851,952, filed Oct. 16, 2006; the contents of all above-named applications are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to novel processing enzymes comprising an alkaline protein tag derived from thermophillic bacteria.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
In accordance with 37 C.F.R. §1.52(e)(5), Applicants enclose herewith the Sequence Listing for the above-captioned application entitled “SEQUENCE LISTING”, created on May 14, 2012. The Sequence Listing is made up of 48 kilobytes, and the information contained in the attached “SEQUENCE LISTING” is identical to the information in the specification as originally filed. No new matter is added.
BACKGROUND OF THE INVENTION
Expression of recombinant proteins in microbial host cells is widely used for industrial production of human therapeutic proteins. A fusion partner may be attached to the N-terminal (or C-terminal) of the target protein and serves to increase expression level, solubility or correct folding of the target protein and to facilitate easy purification during downstream processing. Removal of the fusion partner from the target protein is mostly necessary in order to conserve the biological activity of the target protein. The processing of the fusion protein can either be performed after expression or after one or more initial purification steps using suitable proteases capable of cleaving the fusion partner to release the target protein. The recombinant target protein with or without a fusion partner may also need to be modified after translation to obtain certain biological features using one or more enzymatic steps. Such modification includes removal of unwanted glycosylations with mannose, fucose or xylose sugars or introductions of glycosylations. Modifications may also include deamidations, amidations, methylations, phosphorylations, dephosphorylations, sulfations, acetylations or transaminations.
To decrease the cost of the down stream processing steps it is pivotal that these processing enzymes are cheap to produce e.g. can be expressed in high amounts and purified with a small number of chromatographic steps. Furthermore it is desirable that processing enzymes used for cleavage of fusion proteins can be easily removed after processing of the target protein. It may also be an advantage that the processing enzymes can be immobilised to purifications columns.
The present invention provides novel processing enzymes which are well suited for modification of target proteins including cleavage of fusion partners from a fusion protein. Furthermore, the novel processing enzymes can easily be separated from the target protein after the processing step.
SUMMARY OF THE INVENTION
In one aspect the invention is related to processing enzymes comprising an N-terminally attached tag derived from highly basic ribosomal proteins from thermophilic bacteria.
Some of these tags are disclosed in copending patent application PCT/EP2006/061493.
The tag will facilitate expression of soluble tagged processing enzyme in high yields. The tag will also enable high purity after a single cation exchange chromatography purification step as well as easy removal of the tagged processing enzyme following processing of the target protein precursor. The tags also enable on-column processing using cation exchange chromatography matrix as immobilizer for the tagged processing enzyme.
With the expression “a highly basic protein” is meant a protein having a high percent of the basic amino acid residues Lys and Arg, e.g. at least about 15% of the total number of amino acid residues in the protein.
With “ribosomal proteins” is meant peptide or polypeptide subunits of the ribosome which are the particles that catalyze mRNA-directed protein synthesis in all organisms. Ribosomal proteins are defined on the basis of their sequence by ribosomal signatures as reported in domain databases such as InterPro and Prosite. The advantage of using ribosomal proteins is that they are mostly very positively charged.
In the present context “thermophilic microorganisms” means organisms which grow optimally at about 50° C. to about 100° C. This is in contrast to mesophiles which in general grow optimally at temperatures from 30-37° C. The term “thermophilic bacteria” will in this context cover hyperthermophilic bacteria as well.
In one embodiment the tag contains no cysteine residues.
In a further embodiment the tag comprises from about 15 to about 250, from about 15 to about 225, from 15 to about 200, from about 15 to about 175, from about 15 to about 150, from 15 to about 75, or from about 15 to about 50 amino acid residues.
In one embodiment the tag has from about 15 to about 250 amino acid residues.
In one embodiment the tag has from about 15 to about 200 amino acid residues.
In one embodiment the tag has from about 15 to about 150 amino acid residues.
In one embodiment the tag has from about 15 to about 100 amino acid residues.
In one embodiment the tag has from about 15 to about 50 amino acid residues.
In a further embodiment the tag comprises from about 20 to about 120, from about 20 to about 100, from about 20 to about 90, from about 20 to about 75 amino acid residues or from about 20 to about 50 amino acid residues.
In one embodiment the tag has from about 20 to about 120 amino acid residues.
In one embodiment the tag has from about 20 to about 100 amino acid residues.
In one embodiment the tag has from about 20 to about 90 amino acid residues.
In one embodiment the tag has from about 20 to about 75 amino acid residues.
In one embodiment the tag has from about 20 to about 50 amino acid residues.
In one embodiment the tag has from about 50 to about 250 amino acid residues.
In one embodiment the tag has from about 50 to about 225 amino acid residues.
In one embodiment the tag has from about 50 to about 200 amino acid residues.
In one embodiment the tag has from about 50 to about 175 amino acid residues.
In one embodiment the tag has from about 50 to about 150 amino acid residues.
In one embodiment the tag has from about 50 to about 125 amino acid residues.
In one embodiment the tag has from about 50 to about 100 amino acid residues.
In one embodiment the tag has from about 50 to about 75 amino acid residues.
In one embodiment the tag has from about 75 to about 250 amino acid residues.
In one embodiment the tag has from about 75 to about 225 amino acid residues.
In one embodiment the tag has from about 75 to about 200 amino acid residues.
In one embodiment the tag has from about 75 to about 175 amino acid residues.
In one embodiment the tag has from about 75 to about 150 amino acid residues.
In one embodiment the tag has from about 75 to about 125 amino acid residues.
In one embodiment the tag has from about 75 to about 100 amino acid residues.
In one embodiment the tag has from about 100 to about 250 amino acid residues.
In one embodiment the tag has from about 100 to about 225 amino acid residues.
In one embodiment the tag has from about 100 to about 200 amino acid residues.
In one embodiment the tag has from about 100 to about 175 amino acid residues.
In one embodiment the tag has from about 100 to about 150 amino acid residues.
In one embodiment the tag has from about 100 to about 125 amino acid residues.
The tag will typically contain from at least about 15% basic amino acid residues and the purification tag may contain from about 15 to about 50%, from about 15 to about 45%, from about 15 to about 40, from about 15 to about 35%, from about 15 to about 30%, from about 15 to about 25% or from about 15 to about 20% basic amino acid residues.
In another embodiment the tag comprises from about 20 to about 50%, from about 20 to about 40% or from about 20 to about 30 basic amino acid residues.
In another embodiment the tag comprises from about 40% to about 50% basic amino acid residues.
In another embodiment the tag comprises from about 40% to about 60% basic amino acid residues, Lys and Arg.
In one embodiment the tag comprises from 15 to 50% basic amino acid residues.
In another embodiment the tag comprises from 15 to 45% basic amino acid residues.
In another embodiment the tag comprises from 15 to 40% basic amino acid residues.
In another embodiment the tag comprises from 15 to 35% basic amino acid residues.
In another embodiment the tag comprises from 15 to 30% basic amino acid residues.
In another embodiment the tag comprises from 15 to 25% basic amino acid residues.
In another embodiment the tag comprises from 15 to 20% basic amino acid residues.
In one embodiment the tags are derived from the a ribosomal protein L9 family, which is one of the proteins of the large ribosomal subunit, as described in for example, the Prosite data base (Hulo N., Bairoch A., Bulliard V., Cerutti L., De Castro E., Langendijk-Genevaux P. S., Pagni M., Sigrist C. J. A. “The PROSITE database.” Nucleic Acids Res. 34:D227-D230 (2006).
In one embodiment of the invention the tag is selected from the group of peptide sequences consisting of







(SEQ ID NO: 1)


MSKTIVRKNESIDDALRRFKRAVSKTGTLQEVRKREFYEKPSVRRKKKSE


AARKRK;


 


(SEQ ID NO: 2)


MGKKTVGVKKRLAKAYKQNRRAPVWITVKTKRSVFGSPKRRHWRRSKLKV;


 


(SEQ ID NO: 3)


MKRTYQPSRRKRKRTHGFLARKRTPGGRRVLKNRRRKGRWRLTV;


 


(SEQ ID NO: 4)


MGKGDRRTRRGKIWRGTYGKYRPRKKK;


 


(SEQ ID NO: 5)


MAKVKMKTNRSAAKRFKVTAKGKIKRWKSGGAHYNTKKSSKRKRHLRKHT


YVKDNMLKHVKALLKEF;


 


(SEQ ID NO: 6)


MPKHSKRYLEARKLVDRTKYYDLDEAIELVKKTATAKFDETIELHIQTGI


DYRKPEQHIRGTIVLPHGTGKEVKVLVFAKGEKAKEALEAGADYVGAEDL


VEKIEKEGFLDFDVAIATPDMMRIIGRLGKILGPRGLMPSPKSGTVTQEV


AEAVKEFKKGRIEVRTDKTGNIHIPVGKRSFDNEKLKENIIAAIKQIMQM


KPAGVKGQFIKKWLASTMGPGIKLNLQSLLKE,


 


(SEQ ID NO: 7)


MAQVDLLNVKGEKVGTLEISDFVFNIDPNYDVMWRYVDMQLSNRRAGTAS


TKTRGEVSGGGRKPWPQKHTGRARHGSIRSPIWRHGGWHGPKPRDWSKKL


NKKMKKLALRSALSVKYRENKLLVLDDLKLERPKTKSLKEILQNLQLSDK


KTLIVLPWKEEGYMNVKLSGRNLPDVKVIIADNPNNSKNGEKAVRIDGLN


VFDMLKYDYLVLTRDMVSKIEEVLGNEAGKALTA,


 


(SEQ ID NO: 8)


MRYEYVPLKDQYEKEIVPALMKEFNYKNIHQVPKLVKIVINMGIGEGSRN


YDLIERHANELAKITGQKPIVTRARKSISNFKIRKGMPIGLKVTLRGARM


YNFLYKLINIVLPKVRDFRGLDPNSFDGRGNYSFGLSEQLVFPELNPDEV


RRIQGMDITIVTTAKTDQEARRLLELFGMPFKRG,


 


(SEQ ID NO: 9)


MSRLAKKPIVLPQGVTVEIKDNVVKVKGPKGELSQEFLPYVKIEVEGNEV


WVRPNEEQIIRKSDWRKVKMFQGTYWSLIRNMWGVTEGYKKELEIVGIGY


RAQLQGNTLVMNLGYAHPWYEIPSDVKIEVPAPNRIIVSGIDKQRVGQVA


AEIRAFRPPNVYTGKGIRYVGEWRQKEGKKA,


 


(SEQ ID NO: 10)


MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHE


KEIEKRKKEREREESEKILKELKKRTHWKVKAGEGGKIFGAVTAATVAEE


ISKTTGLKLDKRWFKLDKPIKELGEYSLEVSLPGGVKDTIKIRVEREE,


 


(SEQ ID NO: 11)


MLTRQQKELIVKEMSEIFKKTSLILFADFLGFTVADLTELRSRLREKYGD


GARFRVVKNTLLNLALKNAEYEGYEEFLKGPTAVLYVTEGDPVEAVKIIY


NFYKDKKADLSRLKGGFLEGKKFTAEEVENIAKLPSKEELYAMLVGRVKA


PITGLVFALSGILRNLVYVLNAIKEKKSE,


 


(SEQ ID NO: 12)


MARYFPVQKTTMIKPEEVERKWYVVDASGKVLGRLATRIAKILMGKHKPN


YTPHVDTGDYVIWNADKVVLTGKKLDQKVYYWHSGYPGGLKSLTARQMLE


KHPERLIWLAVKRMLPKNRKGRKMLKRLKVYASPEHPHQAQKPEPIEL,


 


(SEQ ID NO: 13)


MRLEDLRPTPGAMKKRKRVGRGPGSGHGKTSGRGHKGQKARGSGKVHIWF


EGGQTPLQRRLPKRGFKNINKKVYAVVNVKVLEERFEANEEVTPEKLIER


KIIKDLKDGVKILGDGELTKPLVVKAHAFSKSAVEKIESAGGKAEVI,


 


(SEQ ID NO: 14)


MRHRVKRHKLGRYGSHRKSLLRNLSREIVEHGSIVTTTAKAKALKTFMDK


LVSKAIEAATTDDRARSVHLRRQINAVLGDRRLTNKLVDEIAKNYVGRRG


GYVRVLRIGFRRGDAAEMSLVQLVEASSQEG,


 


(SEQ ID NO: 15)


MDHLVKIIEKKYEKKEIPDFRPGDTVRVHVKVIEGDRERTQVFEGIVIAK


RGSGINKTFTVRRIGSHGVGVERIFPVHSPVVEKIEVVRKGKVRRAKLYY


LRNVRGKIRIKERRD,


 


(SEQ ID NO: 16)


MRVKRAVHAKKKRKKYLKAAKGYRGALSRRYKLAKQMYVRSKWYSYVGRK


QKKRDMRKLWITRINIAARNEGLKYSELIHGLKLAGVSINRKMLSELAVN


DPEAFKEYVKIAKEALAS,


 


(SEQ ID NO: 17)


MLYAIVETAGRQYRVEEGKILYTEKQKDYSPGDEIVFDRWFVRKDGEVLV


GKPYVEGAKVVGKVLEHAKARKVKTVKYRPRKNSKVEKGHRQWYTAIKIE


KIEL,


 


(SEQ ID NO: 18)


MKQEKLSLHDVLIRPIITEKALILREQRKYVFEVNPLANKNLVKEAVEKL


FNVKVEKVNILNMKPKPKRRGIFEGKTRSWKKAWTLKEGYTIKELEGEH,


 


(SEQ ID NO: 19)


MAHKKSGGVAKNGRDSLPKYLGVKVGDGQIVKAGNILVRQRGTRFYPGKN


VGMGRDFTLFALKDGRVKFETKNNKKYVSVYEE,


 


(SEQ ID NO: 20)


MKASELRNYTDEELKNLLEEKKRQLMELRFQLAMGQLKNTSLIKLTKRDI


ARIKTILRERELGIRR,


 


(SEQ ID NO: 21)


MPKKLKIKLVKSPIGYSWDQKDTVKRLGLKKLNQVVIKDDLPQIRGMIRK


VKHLVEVEEIEEGGSNA,


 


(SEQ ID NO: 22)


MPKVKTNRSAAKRFRITKNGKIMRNHAYRSHKTGKKRRNALRALRKKDVV


SSADKNRVLRLLGKK,


 


(SEQ ID NO: 23)


MGQKVHPRGFRLGLSADWQAKWFNEKNYKEWLLEDEEIRKIIKNKYYHAG


ISEIYVERPDAERINITVKTARPGIIIGRKGSEITSLREELERKFNRRWI


NIEEIKTPELDAQLVAESIASRIEKRASYKVAMKRAIMNAMRKGAQGIKV


MVAGRLGGAEIARREWYLRGRLPLQKIKAIIDYGTATAWTKYGTIGIKVW


IYKGDADI,


 


(SEQ ID NO: 24)


METQGVMKEIQYEEFEEKIIEIRRTSKVTKGGKNLSFRVVAIVGNKNGKV


GLGIGKAREVPEAIRKAISAAKRNIVEVPVINGTIPHEVIGRQDASKVLL


KPAAPGTGIIAGGTVRAVVELAGIQNILTKSLGSTNPLNLALATMNGLKN


LLDPRKVAKLRDISVEEVFKGVRRENNA,


 


(SEQ ID NO: 25)


MVSLDPEKKNEIIKEFQIHENDTGSVEVQIALLTARIKHLTEHLRKHPKD


FHSRRGLMKMIGRRRKMLKYLRHKKPEVYRELIAKLGIRK,


 


(SEQ ID NO: 26)


MGRSRKKGPYVDRKLLEKIRKLNETGEKKVIKTWSRASMIIPEMVGHTIA


VYNGMKHIPVYITENMIGHRLGEFAPTRRFGGHADKKAKKGELKK


and


 


(SEQ ID: NO 27)


MPNIKSAKKRVRVSEKRRLRNKAYKTFFKNRIKEVLKAIENKEPKEWLEL


TRKAQAAIDKAVSKGVIHKNQGARRKARLFEKVNEYLRTLETTQE.






In another embodiment the tag is selected from the group consisting of







(SEQ ID NO: 18)


MKQEKLSLHDVLIRPIITEKALILREQRKYVFEVNPLANKNLVKEAVEKL


FNVKVEKVNILNMKPKPKRRGIFEGKTRSWKKAWTLKEGYTIKELEGEH


and


 


(SEQ ID NO: 19)


MAHKKSGGVAKNGRDSLPKYLGVKVGDGQIVKAGNILVRQRGTRFYPGKN


VGMGRDFTLFALKDGRVKFETKNNKKYVSVYEE.






In another embodiment the tag is selected from the group consisting of







(SEQ ID NO: 10)


MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHE


KEIEKRKKEREREESEKILKELKKRTHVVKVKAGEGGKIFGAVTAATVAE


EISKTTGLKLDKRWFKLDKPIKELGEYSLEVSLPGGVKDTIKIRVEREE;


 


(SEQ ID NO: 15)


MDHLVKIIEKKYEKKEIPDFRPGDTVRVHVKVIEGDRERTQVFEGIVIAK


RGSGINKTFTVRRIGSHGVGVERIFPVHSPVVEKIEVVRKGKVRRAKLYY


LRNVRGKIRIKERRD


and


 


(SEQ ID NO: 1)


MSKTIVRKNESIDDALRRFKRAVSKTGTLQEVRKREFYEKPSVRRKKKSE


AARKRK.






In another embodiment the tag is selected from the group consisting of







(SEQ ID NO: 6)


MPKHSKRYLEARKLVDRTKYYDLDEAIELVKKTATAKFDETIELHIQTGI


DYRKPEQHIRGTIVLPHGTGKEVKVLVFAKGEKAKEALEAGADYVGAEDL


VEKIEKEGFLDFDVAIATPDMMRIIGRLGKILGPRGLMPSPKSGTVTQEV


AEAVKEFKKGRIEVRTDKTGNIHIPVGKRSFDNEKLKENIIAAIKQIMQM


KPAGVKGQFIKKVVLASTMGPGIKLNLQSLLKE;


 


(SEQ ID NO: 7)


MAQVDLLNVKGEKVGTLEISDFVFNIDPNYDVMWRYVDMQLSNRRAGTAS


TKTRGEVSGGGRKPWPQKHTGRARHGSIRSPIWRHGGVVHGPKPRDWSKK


LNKKMKKLALRSALSVKYRENKLLVLDDLKLERPKTKSLKEILQNLQLSD


KKTLIVLPWKEEGYMNVKLSGRNLPDVKVIIADNPNNSKNGEKAVRIDGL


NVFDMLKYDYLVLTRDMVSKIEEVLGNEAGKALTA,


 


(SEQ ID NO: 9)


MSRLAKKPIVLPQGVTVEIKDNVVKVKGPKGELSQEFLPYVKIEVEGNEV


WVRPNEEQIIRKSDWRKVKMFQGTYWSLIRNMVVGVTEGYKKELEIVGIG


YRAQLQGNTLVMNLGYAHPVVYEIPSDVKIEVPAPNRIIVSGIDKQRVGQ


VAAEIRAFRPPNVYTGKGIRYVGEWRQKEGKKA,


 


(SEQ ID NO: 23)


MGQKVHPRGFRLGLSADWQAKWFNEKNYKEWLLEDEEIRKIIKNKYYHAG


ISEIYVERPDAERINITVKTARPGIIIGRKGSEITSLREELERKFNRRVV


INIEEIKTPELDAQLVAESIASRIEKRASYKVAMKRAIMNAMRKGAQGIK


VMVAGRLGGAEIARREWYLRGRLPLQKIKAIIDYGTATAWTKYGTIGIKV


WIYKGDADI


and


 


(SEQ ID NO: 24)


METQGVMKEIQYEEFEEKIIEIRRTSKVTKGGKNLSFRVVAIVGNKNGKV


GLGIGKAREVPEAIRKAISAAKRNIVEVPVINGTIPHEVIGRQDASKVLL


KPAAPGTGIIAGGTVRAVVELAGIQNILTKSLGSTNPLNLALATMNGLKN


LLDPRKVAKLRDISVEEVFKGVRRENNA,






In another embodiment the tag is selected from the group consisting of







(SEQ ID NO: 10)


MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHE


KEIEKRKKEREREESEKILKELKKRTHVVKVKAGEGGKIFGAVTAATVAE


EISKTTGLKLDKRWFKLDKPIKELGEYSLEVSLPGGVKDTIKIRVEREE;


 


(SEQ ID NO: 41)


MKVILLEPLENLGDVGQVVDVKPGYARNYLLPRGLAVLATESNLKALEAR


IRAQAKRLAERKAEAERLKEILENLTLTIPVRAGETKIYGSVTAKDIAEA


LSRQHGITIDPKRLALEKPIKELGEYVLTYKPHPEVPIQLKVSWAQE;


 


(SEQ ID NO: 42)


MKVIFLKDVKGKGKKGEIKDVADGYANNFLFKQGLAIEATPANIKALEAQ


KQKEQRQAAEELANAKKLKEELEKLTVEIPAKAGEGGRLFGSITSKQIAE


ALQAQHGLKLDKRKIELADAIRSLGYTNVPVKLHPEVTATLKVHVKEQK;


 


(SEQ ID NO: 43)


MKVVLLKDVSKIGKKGEIKNVSDGYARNYLIPKGLALEATPRVLKRLEAE


KRKKEEEKIQIKTQNEELLKMLKKFLYKIPVKAGESGKLFGALTNSDIAK


AVEKIADVNIDKKFIVLEKPIKEIGMYDVLVRLPEGVSGKIKVEVIQEGKN


and


 


(SEQ ID NO: 44)


MKLILTQEVAGLGGPGDVVEVRDGYGRNYLLPKRLAMPASPGAVKQVALI


KRAREVREIRDLDQARALRDQLEALPVTLPARAGSGGRLFGSVTPDDIAA


AVHAAGGPKLDKRRIEISGPIKTIGSHQVTVRLHPEVSATVSVEWPAS.






In a further embodiment the tag has the sequence MKQEKLSLHDVLIRPIITEKALIL







REQRKYVFEVNPLANKNLVKEAVEKLFNVKVEKVNILNMKPKPKRRGIFEG


 


KTRSWKKAVVTLKEGYTIKELEGEH  (SEQ ID NO: 18).






In a further embodiment the tag has the sequence MAHKKSGGVAKNGRDSLP







KYLGVKVGDGQIVKAGNILVRQRGTRFYPGKNVGMGRDFTLFALKDGRVKF


 


ETKNNKKYVSVYEE (SEQ ID NO: 19).






In a further embodiment the tag has the sequence MDHLVKIIEKKYEKKEIPD







FRPGDTVRVHVKVIEGDRERTQVFEGIVIAKRGSGINKTFTVRRIGSHGVG


 


VERIFPVHSPVVEKIEVVRKGKVRRAKLYYLRNVRGKIRIKERRD (SEQ


 


ID NO: 15).






In a further embodiment the tag has the sequence







MKVILLRDVPKIGKKGEIKEVSDGYARNYLIPRGFAKEYTEGLERAIKHEK


 


EIEKRKKEREREESEKILKELKKRTHVVKVKAGEGGKIFGAVTAATVAEEI


 


SKTTGLKLDKRWFKLDKPIKELGEYSLEVSLPGGVKDTIKIRVEREE 


 


(SEQ ID NO: 10).






In a further embodiment the tag has the sequence







MKVILLEPLENLGDVGQVVDVKPGYARNYLLPRGLAVLATESNLKALEARI


 


RAQAKRLAERKAEAERLKEILENLTLTIPVRAGETKIYGSVTAKDIAEALS


 


RQHGITIDPKRLALEKPIKELGEYVLTYKPHPEVPIQLKVSVVAQE (SEQ


 


ID NO: 41).






In a further embodiment the tag has the sequence







MKVIFLKDVKGKGKKGEIKDVADGYANNFLFKQGLAIEATPANIKALEAQK


 


QKEQRQAAEELANAKKLKEELEKLTVEIPAKAGEGGRLFGSITSKQIAEAL


 


QAQHGLKLDKRKIELADAIRSLGYTNVPVKLHPEVTATLKVHVKEQK 


 


(SEQ ID NO: 42).






In a further embodiment the tag has the sequence MKWLLKDVS KIGKKGEIKN







VSDGYARNYLIPKGLALEATPRVLKRLEAEKRKKEEEKIQIKTQNEELLKM


 


LKKFLYKIPVKAGESGKLFGALTNSDIAKAVEKIADVNIDKKFIVLEKPIK


 


EIGMYDVLVRLPEGVSGKIKVEVIQEGKN (SEQ ID NO: 43).






In a further embodiment the tag has the sequence







MKLILTQEVAGLGGPGDVVEVRDGYGRNYLLPKRLAMPASPGAVKQVALIK


 


RAREVREIRDLDQARALRDQLEALPVTLPARAGSGGRLFGSVTPDDIAAAV


 


HAAGGPKLDKRRIEISGPIKTIGSHQVTVRLHPEVSATVSVEWPAS (SEQ


 


ID NO: 44).






The tag may be attached directly to the N-terminal amino acid in the processing enzyme in question or via a linker group. Such linker group may have a multipurpose function i.e. to facilitate attachment of the tag to the processing enzyme and to positionate the tag and the processing enzyme relatively to each other to enable correct folding of the processing enzyme. The linker may also serve to present the tag in a favorable way to the purification matrix used for purification of the tagged processing enzyme.
The linker may have from 1-30, from 1-25, from 1-20, from 1-15, from 1-14, from 1-13, from 1-12, from 1-11, from 1-10, from 1-9, from 1-8, from 1-7, from 1-6, from 1-5 amino, from 1-4 or from 1-3 acid residues.
In a further embodiment the linker will have from 2-30, from 2-25, from 2-20, from 2-15, from 2-14, from 2-13, from 2-12, from 2-11, from 2-10, from 2-9, from 2-8, from 2-7, from 2-6, from 2-5 or from 2-4 amino acid residues.
In a further embodiment the linker will have from 3-30, from 3-25, from 3-20, from 3-15, from 3-14, from 3-13, from 3-12, from 3-11, from 3-10, from 3-9, from 3-8, from 3-7, from 3-6, from 3-5 or from 3-4 amino acid residues.
In a further embodiment the linker will have from 4-30, from 4-25, from 4-20, from 4-15, from 4-14, from 4-13, from 4-12, from 4-11, from 4-10, from 4-9, from 4-8, from 4-7, from 4-6 or from 4-5 amino acid residues.
In a further embodiment the linker will have from 5-30, from 5-25, from 5-20, from 5-15, from 5-14, from 5-13, from 5-12, from 5-11, from 5-10, from 5-9, from 5-8, from 5-7 or from 5-6 amino acid residues.
In a further embodiment the linker will have from 6-30, from 6-25, from 6-20, from 6-15, from 6-14, from 6-13, from 6-12, from 6-11, from 6-10, from 6-9, from 6-8 or from 6-7 amino acid residues.
In a further embodiment the linker will have from 7-30, from 7-25, from 7-20, from 7-15, from 7-14, from 7-13, from 7-12, from 7-11, from 7-10, from 7-9 or from 6-8 amino acid residues.
On one embodiment the linker has 20 amino acid residues.
In another embodiment the linker has 15 amino acid residues.
In another embodiment the linker has 12 amino acid residues.
In another embodiment the linker has 10 amino acid residues.
In another embodiment the linker has 9 amino acid residues.
In another embodiment the linker has 8 amino acid residues.
In another embodiment the linker has 7 amino acid residues.
In another embodiment the linker has 6 amino acid residues.
In another embodiment the linker has 5 amino acid residues.
The amino acid residues in the linker may be any amino acid residue and may be the same or different depending on the intended function of the linker.
In one embodiment the linker will comprise amino acid residues which render the linker a flexible structure such as alternating Ser and Gly residues. Other amino acid residues result in a rigid structure i.e. amino acids which may form alpha helical structures, such as Ala and Leu. Furthermore, certain amino acid residues add a structural bend to the linker e.g. Pro.
Non-limiting examples of linker groups are SSSGSSGSSGSS (SEQ ID NO;28), GGSSGGSS (SEQ ID NO:29), SSSGSGSG (SEQ ID NO:30), ALALALA (SEQ ID NO:31), ALALALAPA (SEQ ID NO:32), SSSALALALA (ID NO:33); SGSGSGSGS (ID NO:34), SSSGSGSGSG (SEQ ID NO:47) and GSSGSGS (SEQ ID NO:48).
In one embodiment the linker has the sequence SSSGSSGSSGSS (SEQ ID NO;28).
In another embodiment the linker has the sequence GGSSGGSS (SEQ ID NO:29).
In another embodiment the linker has the sequence SSSGSGSG (SEQ ID NO:30).
In another embodiment the linker has the sequence ALALALA (SEQ ID NO:31).
In another embodiment the linker has the sequence ALALALAPA (SEQ ID NO:32)
In another embodiment the linker has the sequence SSSALALALA (ID NO:33).
In another embodiment the linker has the sequence SGSGSGSGS (ID NO:34).
In another embodiment the linker has the sequence SSSGSGSGSG (SEQ ID NO:47).
In another embodiment the linker has the sequence GSSGSGS (SEQ ID NO:48).
In one embodiment the above described tags may be a combination of two tags which may be the same or different. Also the tag may be an analogue of the specifically disclosed tags. With an “analogue” is meant a sequence having up to 10% mutations compared to the original sequence. Such mutations will comprise additions, deletions or additions or any combination thereof as long as such mutations do not alter the original functionality of the tag.
In one embodiment the invention encompasses tagged processing enzymes wherein the tag is a truncated analogue of the original tag. The expression “truncated analogue” is meant to cover a sequence of the original tag wherein up to 10% of the original amino acid residues from either the N-terminal or the C-terminal end have been deleted provided that the truncated analogue has retained the functionality of the unmodified sequence.
If the tag comprises a combination of two tags these tags will in one embodiment be connected by a linker.
In one embodiment the linker connecting two tags are selected from the group consisting of SSSGSSGSSGSS (SEQ ID NO;28); GGSSGGSS (SEQ ID NO:29), SSSGSGSG (SEQ ID NO:30), ALALALA (SEQ ID NO:31), ALALALAPA (SEQ ID NO:32), SSSALALALA (ID NO:33); SGSGSGSGS (ID NO:34), SSSGSGSGSG (SEQ ID NO:47) and GSSGSGS (SEQ ID NO:48).
The invention will cover any combination of the embodiments of the tag, the linker and the processing enzyme as disclosed herein.
In one embodiment the tagged processing enzyme will comprise an N-terminal extension comprising a combination of the tag and the linker selected from the following group: SEQ ID NO:1 and SEQ ID NO:28, SEQ ID NO:3 and SEQ ID NO:28, SEQ ID NO:6 and SEQ ID NO:28, SEQ ID NO:9 and SEQ ID NO:28, SEQ ID NO:10 and SEQ ID NO:28, SEQ ID NO:11 and SEQ ID NO:28, SEQ ID NO:12 and SEQ ID NO:28, SEQ ID NO:13 and SEQ ID NO:28, SEQ ID NO:15 and SEQ ID NO:28, SEQ ID NO:16 and SEQ ID NO:28, SEQ ID NO:20 and SEQ ID NO:28, SEQ ID NO:21 and SEQ ID NO:28, SEQ ID NO:24 and SEQ ID NO:28, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:18 and SEQ ID NO:28, SEQ ID NO:19 and SEQ ID NO:28, SEQ ID NO:41 and SEQ ID NO:28, SEQ ID NO:42 and SEQ ID NO:28, SEQ ID NO:43 and SEQ ID NO:28, and SEQ ID NO:44 and SEQ ID NO:28,
In one embodiment the tagged processing enzyme will comprise an N-terminal extension comprising a combination of the tag and the linker selected from the following group: SEQ ID NO:1 and SEQ ID NO:47, SEQ ID NO:3 and SEQ ID NO:47, SEQ ID NO:6 and SEQ ID NO:47, SEQ ID NO:9 and SEQ ID NO:47, SEQ ID NO:10 and SEQ ID NO:47, SEQ ID NO:11 and SEQ ID NO:47, SEQ ID NO:12 and SEQ ID NO:47, SEQ ID NO:13 and SEQ ID NO:47, SEQ ID NO:15 and SEQ ID NO:47, SEQ ID NO:16 and SEQ ID NO:47, SEQ ID NO:20 and SEQ ID NO:47, SEQ ID NO:21 and SEQ ID NO:47, SEQ ID NO:24 and SEQ ID NO:47, SEQ ID NO:27 and SEQ ID NO:47, SEQ ID NO:18 and SEQ ID NO:47, SEQ ID NO:19 and SEQ ID NO:47, SEQ ID NO:41 and SEQ ID NO:47, SEQ ID NO:42 and SEQ ID NO:47, SEQ ID NO:43 and SEQ ID NO:47 and SEQ ID NO:44 and SEQ ID NO:47.
In one embodiment the tagged processing enzyme will comprise an N-terminal extension comprising a combination of the tag and the linker selected from the following group: SEQ ID NO:1 and SEQ ID NO:48, SEQ ID NO:3 and SEQ ID NO:48, SEQ ID NO:6 and SEQ ID NO:48, SEQ ID NO:9 and SEQ ID NO:48, SEQ ID NO:10 and SEQ ID NO:48, SEQ ID NO:11 and SEQ ID NO:48, SEQ ID NO:12 and SEQ ID NO:48, SEQ ID NO:13 and SEQ ID NO:48, SEQ ID NO:15 and SEQ ID NO:48, SEQ ID NO:16 and SEQ ID NO:48, SEQ ID NO:20 and SEQ ID NO:48, SEQ ID NO:21 and SEQ ID NO:48, SEQ ID NO:24 and SEQ ID NO:48, SEQ ID NO:27 and SEQ ID NO:48, SEQ ID NO:18 and SEQ ID NO:48, SEQ ID NO:19 and SEQ ID NO:48, SEQ ID NO:41 and SEQ ID NO:48, SEQ ID NO:42 and SEQ ID NO:48, SEQ ID NO:43 and SEQ ID NO:48 and SEQ ID NO:44 and SEQ ID NO:48.
In on embodiment tag is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 and the linker is selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:47 and SEQ ID NO:48.
In another aspect the present invention is related to a method for making a tagged processing enzyme, which method comprises i) expression of a processing enzyme comprising an N-terminal tag according to any of the herein disclosed embodiments derived from a highly basic protein from a thermophilic bacteria in a suitable expression host, ii) loading the expressed tagged processing enzyme on a cation-exchange column, iii) and eluting the tagged processing enzyme with a suitable eluent.
In another aspect the invention is related to a method for modifying target protein precursors comprising the steps of reacting a tagged processing enzyme according to the invention with the target protein precursor and separating a target protein from the processing enzyme.
The processing enzymes are typically selected from oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
In one embodiment the processing enzyme is a protease which is used to cleave fusion proteins.
In one embodiment the processing protease is selected from the group consisting of aspartic, metallo, serine and cysteine proteases.
In a further embodiment serine proteases include trypsins, chymotrypsins, enterokinase (EK), thrombin, factor Xa and kallikreins.
In one embodiment the cystein proteases are selected from the group of 3C proteases and 2A proteases encoded by the viral genomes of the group of Corona viridae, Picorna viridae, Retro viridae, Adeno viridae or Flavi viridae. Other preferred embodiments include 3C and 2A proteases from Picorna viridae and Corona viridae. 
In a further embodiment the 3C proteases are selected from the Human Rhino Virus 3C protease with the sequence GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTH-AQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVV HSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNG RQGFSAQLKKQYFVEKQ (SEQ ID NO: 35); Hepatitis A strain 18 3C protease with the







sequence STLEIAGLVRKNLVQFGVGEKNGCVRVVVMNALGVKDDWLLV


 


PSHAYKFE-KDYEMMEFYFNRGGTYYSISAGNVVIQSLDVGFQDVVLMKVP


 


TIPKFRDITEHFIKKGDVPRALNRLATLVTTVNGTPMLISEGPLKMEEKAT


 


YVHKKNDGTTVDLTVDQAWRGKGEGLPGMCGGALVSSNQSIQNAILGIHVA


 


GGNSILVAKLVTQEMFQNIDKKIESQ (SEQ ID NO: 45) or






Rabbit hemorrhagic disease virus 3C-like protease with the sequence








GLPGFMRHNGSGWMIHIGNGLYISNTHTARSSCSEIVTCSPTTDLCLVKGE


 


AIRSVAQIAEGTPVCDWKKSPISTYGIKKTLSDSTKIDVLAYDGCTQTTHG


 


DCGLPLYDSSGKIVAIHTGKLLGFSKMCTLIDLTITKGVYE  (SEQ ID


 


NO: 46).






In a further embodiment the protease is from a plant virus. The proteases will typically be from the following plant virus families: Bromoviridae, Partitiviridae, Geminiviridae, Potyviridae, Tombusviridae, Comoviridae, Rhabdoviridae, Reoviridae, Potyviridae, Sequiviridae and Bunyaviridae.
In one embodiment the protease from a plant virus is the mature Tobacco etch virus (TEV) protease with the amino acid sequence:







GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRN


 


NGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFRE


 


PQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGS


 


PLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRL


 


NADSVLWGGHKVFMVKPEEPFQPVKEATQLMN (SEQ ID NO: 38)






The target protein which can be produced from a precursor protein modified by the processing enzyme according to the invention may be selected from human proteins and their analogues such as aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin analogues, human or bovine growth hormone, interleukin, glucagon, GLP-1, GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor α or β, platelet-derived growth factor, GRF (growth hormone releasing factor), immunoglubolines, EPO, TPA, protein C, blood coagulation factors such as FVII, FVIII, FIV and FXIII, exendin-3, exentidin-4, and enzymes or functional analogues thereof.
In a further aspect of the invention the target protein is selected from insulin antagonist or agonist peptides such as, S661 or peptide hormones such as amylin or functional analogues thereof.
In a further aspect of the invention the target protein is selected from enzymes relevant to any modification of proteins such as transglutaminases, transferases and racemases.
In one embodiment the invention is related to a process for removal of a prodomain from the protransglutaminase (proTGase) using a tagged processing enzyme according to the invention.
In another embodiment the invention is related to a process for cleavage of a protransglutaminase (proTGase) with a tagged HRV14 3C protease, a tagged HAV18 3C protease or a tagged RHDV 3C protease. In this embodiment the tag may be selected from SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44 and the linker may be selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:47 and SEQ ID NO:48.
In another embodiment the invention is related to a process for removal of fusion partners of human growth hormone or analogues thereof with a tagged HRV14 3C protease, a tagged HAV18 3C protease, a tagged TEV protease or a tagged RHDV 3C protease. In this embodiment the tag may be selected from SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44 and the linker may be selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:47 and SEQ ID NO:48.
In another embodiment the invention is related to a process for removal of fusion partners of human amylin or analogues thereof with a tagged HRV14 3C protease, a tagged HAV18 3C protease, a tagged TEV protease or a tagged RHDV 3C protease. In this embodiment the tag may be selected from SEQ ID NO:10, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44 and the linker may be selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:47 and SEQ ID NO:48.



DESCRIPTION OF THE DRAWINGS
FIG. 1: discloses a vector map of the pET11a derived vector encoding the pACSH238 processing enzyme consisting of SEQ ID NO:10 as N-terminal purification tag, SEQ ID NO:28 as linker and SEQ ID NO:35 as the HRV14 3C protease. NdeI, XhoI and BamHI cloning sites are indicated as well lacI coding sequence, ampicillin (bla) coding sequence, pBR322 origin of replication, T7 promoter region, T7 terminator region and
FIG. 2 discloses an extracted and deconvoluted mass spectrum of pACSH294 incubated with pACSH239 overnight at 25 degrees Celcius. At 1:100 enzyme to substrate ratio, the S661 peptide (4802.1 Da) is clearly detected as well as the released tag+linker (10168.4 Da). No intact fusion protein was detected, which indicates that the fusion protein was completely cleaved into to the tag part and the S661 peptide. Y-axis indicates intensity of ions measured. X-axis indicates molecular mass in Dalton (Da).



DESCRIPTION OF THE INVENTION
The present invention provides novel processing enzymes which can be used to modify proteins e.g. to cleave fusion proteins expressed in microbial host cells. Furthermore, the present invention provides a method for efficient expression and purification of such tagged processing enzymes.
The processing enzymes according to the present invention will comprise N-terminally fused tags that facilitate soluble expression of the tagged processing enzyme in high yields and enable an efficient purification using a single cation exchange chromatography step as well as easy removal of the tagged processing enzyme after use.
The tags will increase the solubility and hereby also the stability of the tagged processing enzyme. This is an advantage because poorly soluble enzymes require a greater reaction volume, incubation time and more specific reaction conditions.
The tags will also facilitate the binding to a cation column and thus enable on-column cleavage using cation exchange chromatography matrix as immobilizer for the tagged processing enzyme.
Some of the tags used according to the present invention are disclosed in copending application PCT/EP2006/061493.
These tags are derived from strains of thermophilic bacteria with optimum temperatures ranging from −50° C. to above the boiling point of water. The strains that survive at extremely high temperatures are called hyperthermophiles or thermophiles and have a temperature optimum of 80° C. (176° F.) or higher. Thermophilic bacteria occur naturally in hot springs, hot soils, geothermal vents and other places were high temperature are present. Bacillus stearothermophillus from which RS21_BACST (SEQ ID NO:1) was cloned and used as a tag is for example found to grow above 65° C. in many soils. In order to survive the high temperatures, these organisms have evolved proteins which are more stable than those of mesophiles.
The tags according to the present invention derived from such thermophilic bacteria are in general soluble, highly stable and have a very basic pI due to a large amount of Arg and Lys residues present in the amino acid sequence. The pI of the tags will generally be between 9 and 13.5. In one embodiment the tag has a pI above about 9. In another embodiment the tag has a pI above between about 9 and 12.5. In another embodiment the pI of the tag will be between about 10 and about 12.5 and in a further aspect the pI is about 10.
The solubility is believed to be derived from the generally high surface charge of the proteins.
The processing enzymes according to the present invention may be used to process recombinantly expressed proteins to give a desired modified protein.
The processing enzymes are selected from the following group of enzymes: oxidoreductases, which may catalyze oxidation/reduction reactions, transferases, which may transfer a functional group (eg. phosphate groups), hydrolases which catalyze the hydrolysis of various bonds (including proteases which hydrolyse peptide bonds), lyases, which cleave bonds by means other than hydrolysis and oxidation, isomerase, which catalyze changes within a single molecule, or ligase, which join two molecules through covalent bonds.
These enzymes may either be wildtype enzymes or variants or analogues thereof modified by well known mutagenesis steps.
Examples of tagged proteases that can be used for cleavage of fusion partners from a target protein or removal of prodomains or trimming of amino acids in the N-terminal or C-terminal of target proteins are aspartic, metallo, serine and cysteine proteases.
Non-limiting examples of serine proteases include trypsins, chymotrypsins, enterokinase (EK), thrombin, factor Xa and kallikreins.
Non-limiting examples of cysteine proteases include viral proteases obtained from the group of Corona viridae, Picorna viridae, Retro viridae, Adeno viridae, Calici viridae or Flavi viridae. Further examples include proteases from herpes virus, cytomegalo virus, hepatitis A-D virus, dengue virus, rhino virus, polio virus, coxsackie virus, servere acute respiratory syndrome virus (SARS), mengo virus, polio virus, footh and mouth disease virus, human immune deficiency virus (HIV), human T-lymphotrophic virus (HTLV), human foamy virus and adenovirus. Other non-limiting examples includes cysteine proteases derived from bacteria such as Leishmania sp. or eukaryotic microbes such as Trypanosoma sp.
In one embodiment the cysteine proteases are selected from the group of 3C proteases and 2A proteases encoded by the viral genomes of the group of Corona viridae, Picorna viridae, Retro viridae, Adeno viridae or Flavi viridae. Other preferred embodiments include 3C and 2A proteases from Picorna viridae and Corona viridae. Other examples of cysteine proteases include caspases such as human caspases 1-10.
A review of 3C proteases is found in Krausslich, H. G. & Wimmer, E., Viral proteinases, Annual Review of Biochemistry (1988) 57, 701-754.
Non-limiting examples of metallo proteases include matrix metallo proteases such as matrixins or membrane bound metallo proteases such as MT1-MMP.
Other embodiments includes aspartic proteases such as pepsin and cathepsin e.g. cathepsin D and chymosin.
Other examples of enzymes include lipases, phosphatases, glycosyl hydrolases (eg. mannosidases, xylosidases, fucosidases), kinases, mono or dioxidases, peroxidases, transaminases, carboxypeptidases, amidases, esterases and phosphatases.
The target protein precursor which can be modified by the tagged processing enzyme may be any protein and illustrative examples are aprotinin, tissue factor pathway inhibitor or other protease inhibitors, insulin or insulin precursors, human or bovine growth hormone, interleukin, glucagon, GLP-1, GLP-2, IGF-I, IGF-II, tissue plasminogen activator, transforming growth factor α or β, platelet-derived growth factor, GRF (growth hormone releasing factor), immunoglubolines, EPO, TPA, protein C, blood coagulation factors such as FVII, FVIII, FIV and FXIII, exendin-3, exentidin-4, and enzymes or functional analogues thereof.
Other examples of target proteins are transforming growth factor α (TGF-α), transforming growth factor β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), thrombopoietin (TPO), interferon, pro-urokinase, urokinase, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrandt factor, a cytokine, e.g. an interleukin such as interleukin (IL) 1, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21 IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32 or IL-33, a colony stimulating factor (CFS) such as GM-CSF, stem cell factor, a tumor necrosis factor such as TNF-α, lymphotoxin-α, lymphotoxin-β, CD40L, or CD30L, a protease inhibitor e.g. aprotinin, an enzyme such as superoxide dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase, ribonuclease, catalase, uricase, bilirubin oxidase, trypsin, papain, alkaline phosphatase, β-glucoronidase, purine nucleoside phosphorylase or batroxobin, an opioid, e.g. endorphins, enkephalins or non-natural opioids, a hormone or neuropeptide, e.g. calcitonin, glucagon, gastrins, adrenocorticotropic hormone (ACTH), cholecystokinins, lutenizing hormone, gonadotropin-releasing hormone, chorionic gonadotropin, corticotrophin-releasing factor, vasopressin, oxytocin, antidiuretic hormones, thyroid-stimulating hormone, thyrotropin-releasing hormone, relaxin, prolactin, peptide YY, neuropeptide Y, pancreatic polypeptide, leptin, CART (cocaine and amphetamine regulated transcript), a CART related peptide, perilipin, melanocortins (melanocyte-stimulating hormones) such as MC-4, melanin-concentrating hormones, natriuretic peptides, adrenomedullin, endothelin, secretin, amylin, vasoactive intestinal peptide (VIP), pituary adenylate cyclase activating polypeptide (PACAP), bombesin, bombesin-like peptides, thymosin, heparin-binding protein, soluble CD4, hypothalmic releasing factor and melanotonins.
In another embodiment of the invention the target protein may be insulin receptor agonist or antagonist peptides or other peptides designed to interact with other cell membrane receptors.
One embodiment of the present invention will include co-expression of the tagged processing enzyme according to the invention and the target protein precursor thus facilitating in vivo processing of the target protein precursor and easy removal of the processing enzyme during initial purification steps.
The expressed tagged processing enzyme produced by the method according to the invention may be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, releasing the tagged processing enzyme by mechanical cell disruption, such as ultrasonication or pressure, precipitating the proteineous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate.
Any suitable cation exchange matrix available from commercial suppliers can be used in the method according to the invention and a non limiting list of suitable cation exchange column material is: SP Sepharose Fast Flow, SP Sepharose XL; Streamline SP XL, Streamline Direct CST, Obelix SP, S-Support Unosphere, SP Sepharose High Performance, Source30S and Toyopearl SP650S TosoHaas.
The steps following the processing step e.g. cleavage of a fusion protein may include a cation exchange column purification as in the first step. Purification steps following cleavage may also comprise other means of purification such as anion exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography (see, for example, Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982).
The nucleic acid construct encoding the tagged processing enzyme according to the invention may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the fusion protein by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Labora-tory, Cold Spring Harbor, N.Y., 1989).
The nucleic acid construct encoding the tagged processing enzyme may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesised, e.g. in an automatic DNA synthesiser, purified, annealed, ligated and cloned in suitable vectors. The DNA sequences encoding the fusion protein may also be prepared by polymerase chain reaction such as splicing by overlap extension PCR using specific primers, for instance as described in U.S. Pat. No. 4,683,202, Saiki et al., Science 239 (1988), 487-491, or Sambrook et al., supra.
Furthermore, the nucleic acid construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.
The DNA sequences encoding the tagged processing enzyme are usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the tagged processing enzyme is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, “operably linked” indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the fusion protein.
Expression vectors for use in expressing the tagged processing enzyme will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or the phage Lambda PR or PL promoters or promoters used for expression in E. coli eg. lac, trp, phoA, araBAD, tac, bacteriophage T7 and cspA.
The vector may also comprise a selectable marker, e.g. a gene product which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or a marker gene which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
The DNA sequences encoding the tagged processing enzyme may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) terminators. Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the fusion polypeptide sequence itself. Such RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the insertion site. Examples of polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 EIb region, the human growth hormone gene terminator (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981). The expression vectors may also include a non coding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
The host cell into which the DNA construct encoding the tagged processing enzyme is introduced may be any bacteria cell which is capable of producing the tagged processing enzyme.
Examples of bacterial host cells are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as strains of Escherichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing protein in bacteria such as E. coli, the protein may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the polypeptide is refolded by diluting the denaturing agent. In the latter case, the target protein may be cloned with a strong signal peptide sequence such as phoA, degQ, degS, degP, OmpA, OmpF, OmpH, OmpP, OmpT, lamb or pelB (from Erwania carotovora) and the polypeptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the polypeptide.
The transformed or transfected host cell is then cultured in a suitable nutrient medium under conditions permitting expression of the tagged processing enzyme after which all or part of the resulting peptide may be recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
“Application” means a sample containing the fusion protein which is loaded on a purification column.
“Flow through” means the part of the application containing host cell proteins and contaminants which do not bind to the purification column.
“Main peak” refers to the peak in a purification chromatogram which has the highest UV intensity and which contains the fusion protein.
“mAU” is milliabsorbance units.
“UV 280 intensity” is the absorbance at a wavelength of 280 nm at which proteins will absorb, measured in milliabsorbance units.
“UV214” is the absorbance at a wavelength of 214 nm at which proteins will absorb, measured in milliabsorbance units.
“IPTG” is isopropyl-β-D-thiogalactopyranoside.
“TIC” is Total Ion Count.
“HPLC” is high performance liquid chromatography.
The expression “protein” will cover both peptides and polypeptides.
A “target protein precursor” is a protein which is a precursor for the target protein and which must be processed by the processing enzyme to obtain the target protein. Examples includes fusion protein from which the fusion partner has to be removed by a processing enzyme to obtain the target protein, proteins from which post translational modifications (eg. glycosylations) have to be removed by a processing enzyme to obtain the target protein or proteins to which post translational modifications have to be added by a processing enzyme or combinations of the above.
A “target protein” is a protein which has been processed by a processing enzyme to reach its intended form.
“SOE PCR” means Splicing by overlap extension PCR.
“LC-MS” refers to liquid chromatography mass spectrometry.
“% Solubility” is defined as the amount of soluble protein from host cell lysate divided by amount of soluble+insoluble fusion protein from host cell lysate×100.
“% Purity” is defined as the amount of the protein of interest divided by the amount of protein of interest+the amount of host cell contaminants×100.
SDS-PAGE is sodium dodecyl sulfate polyacrylamide gel electrophoreses.
In the present context the three-letter or one-letter indications of the amino acids have been used in their conventional meaning as indicated in table 1. Unless indicated explicitly, the amino acids mentioned herein are L-amino acids. Further, the left and right ends of an amino acid sequence of a peptide are, respectively, the N- and C-termini unless otherwise specified.







TABLE 1







Abbreviations for amino acids:











Amino acid
Tree-letter code
One-letter code







Glycine
Gly
G



Proline
Pro
P



Alanine
Ala
A



Valine
Val
V



Leucine
Leu
L



Isoleucine
Ile
I



Methionine
Met
M



Cysteine
Cys
C



Phenylalanine
Phe
F



Tyrosine
Tyr
Y



Tryptophan
Trp
W



Histidine
His
H



Lysine
Lys
K



Arginine
Arg
R



Glutamine
Gin
Q



Asparagine
Asn
N



Glutamic Acid
Glu
E



Aspartic Acid
Asp
D



Serine
Ser
S



Threonine
Thr
T










All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).
All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.
This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.
EXAMPLES
Example 1
Synthesis of a HRV14 3C Protease Encoding Sequence Using Overlap PCR
The amino acid sequence of the mature Human Rhino Virus strain 14 (HRV14) 3C protease is as follows:







GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNG


 


QKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLV


 


VHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCAT


 


GKIFGIHVGGNGRQGFSAQLKKQYFVEKQ (SEQ ID NO: 35)






A DNA sequence encoding SEQ ID NO: 35 and codon optimized for expression in E. coli was generated. The mature HRV14 3C protease synthetic gene fragment was obtained by using splicing by overlap extension PCR methodology using two consecutive steps of PCR. In the first PCR reaction, 16 overlapping primers (eight forward and eight reverse primers) were used to generate the entire HRV14 sequence, including a 5-end region encoding a flexible linker (SEQ ID NO:28). Primers were  ̃50 bp of size and the overlap between primers was  ̃20 bp. In the second PCR reaction, terminal forward and reverse primers comprising XhoI and BamHI cloning sites, respectively, were used to amplify the whole sequence using the product of the first PCR reaction as a template. The Phusion PCR system (Finnzymes) was used. The first PCR reaction was setup as follows with suitable amounts of all 16 primers essentially according to the manufactures instructions. The PCR condition for the first reaction was as follows;
98° C. 30 sec (initial denaturation)
98° C. 10 sec (denaturation)
50° C. 30 sec (annealing)
72° C. 15 sec (elongation)
for 10 cycles
72° C. 5 min (final elongation)
The PCR product from the first reaction was purified directly from the PCR solution using the GFXTM PCR DNA and Gel Band Purification Kit (GE Health care).
In the second PCR reaction, the most terminal forward and reverse primers were used to amplify the entire HRV14 3C encoding sequence from the PCR product from the first reaction. The conditions were the same as described for the first reaction, except that 55° C. were used as annealing temperature and 20 cycles were used instead of 10.
The PCR product from the second reaction has a size of  ̃600 base pairs as expected, judged by agarose gel electrophoresis. The PCR band was excised from agarose gels and the PCR product was purified using the GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare) according to the manufacturer's instructions. The purified PCR product was subcloned into plasmids using established TOPO cloning systems (Invitrogen) according to the manufacturer's instructions and ligation products were transformed into TOP10 E. coli cells (Invitrogen) for plasmid probagation. The correct sequence of XhoI/BamHI inserts of plasmids obtained from isolated TOP10 clones was verified by DNA sequencing.
Ligation of the HRV14 Sequence with Different Thermostable Alkaline Protein Tags into pET11a Vectors
The XhoI/BamHI fragment comprising the HRV14 3C protease encoding region was excised from TOPO plasmids vectors (Invitrogen). XhoI/BamHI fragments were then ligated into pET11a vectors (Novagen) so that inserts encoding different N-terminal thermostable alkaline tags were linked to the 5-end of the linker and protease encoding region. Sixteen pET11a vectors encoding different tags fused to the N-terminal of the HRV14 3C protease with an intervening linker was generated as exemplified in FIG. 1.
Verification of the DNA sequence was performed by restriction enzyme cleavage and/or DNA sequencing.
The following tagged enzymes were produced:













Product name
Tag
Linker
Processing enzyme







pACSH240
SEQ ID NO: 1
SEQ ID NO: 28
SEQ ID NO: 35


pACSH329
SEQ ID NO: 3
SEQ ID NO: 28
SEQ ID NO: 35


pACSH319
SEQ ID: NO:6
SEQ ID NO: 28
SEQ ID NO: 35


pACSH320
SEQ ID NO: 8
SEQ ID NO: 28
SEQ ID NO: 35


pACSH321
SEQ ID NO:9
SEQ ID NO: 28
SEQ ID NO: 35


pACSH238
SEQ ID NO: 10
SEQ ID NO: 28
SEQ ID NO: 35


pACSH322
SEQ ID NO: 11
SEQ ID NO: 28
SEQ ID NO: 35


pACSH323
SEQ ID NO: 12
SEQ ID NO: 28
SEQ ID NO: 35


pACSH324
SEQ ID NO: 13
SEQ ID NO: 28
SEQ ID NO: 35


pACSH325
SEQ ID NO: 15 
SEQ ID NO: 28
SEQ ID NO: 35


pACSH326
SEQ ID NO: 16 
SEQ ID NO: 28
SEQ ID NO: 35


pACSH239
SEQ ID NO: 19 
SEQ ID NO: 28
SEQ ID NO: 35


pACSH327
SEQ ID NO: 20
SEQ ID NO: 28
SEQ ID NO: 35


pACSH328
SEQ IDNO: 21
SEQ ID NO: 28
SEQ ID NO: 35


pACSH331
SEQ ID NO: 24
SEQ ID NO: 28
SEQ ID NO: 35


pACSH332
SEQ ID NO: 27
SEQ ID NO: 28
SEQ ID NO: 35









Expression of Tagged HRV14 3C Proteases.

pET 11a vector construct encoding the HRV14 3C protease with different N-terminal tags were transformed into the Rosetta (DE3) (Novagen) E. coli strain by means of heat shock or chemical based standard methods. Transformed cells were probagated on LB (Luria-Bertani) medium agar plates containing 150 micrograms per milliter Ampicillin and 34 micrograms per milliter chloramphinicol overnight. Liquid LB medium with ampecillin and chloramphinicol was inoculated from culture plates and cultivated in shaker flasks at 37 degrees C. and 240 rpm. The temperature was lowered to 30 degrees C. when OD600:  ̃0.4-0.8 was reached. 20-30 minutes after protein induction was initiated by adding 0.5 mM IPTG to the culture medium. The final OD600 obtained after 3 hours induction was in range of 1.6-2.8. Cells were harvested by centrifugation. SDS-PAGE of lysates of pellets containing induced and uninduced cells, as well as soluble and insoluble fractions of induced cells (obtained by ultrasonication and centrifugation in a buffer consisting of 25 mM NaPO4 pH 7) was performed to evaluate expression level and solubility of the HRV14 3C protease variants. Although differences were observed in expression levels, a very high solubility ( ̃80% or more) was observed for most tagged versions of the HRV14 3C protease.
Purification of HRV14 3C Protease N-Terminally Tagged with SEQ ID NO:19(pACSH239):
Cell culture pellets (from 40 ml of culture) of the pACSH239 protease were disrupted by ultrasonication in a buffer containing 25 mM NaPO4 pH 7. Cell debris was spun down by centrifugation at 10.000 g. Supernatants were sterilefiltrated before purification, which was carried out using a AKTA explorer 100 purification system (GE Healthcare). A prepacked SP Sepharose FF HiTrap column with a 5 ml column volume (GE Healthcare) was used for the separation at a flow rate of 3 ml/min using the following buffers:
Buffer A: 50 mM sodium phosphate, pH 7+1 mM EDTA
Buffer B: 50 mM sodium phosphate, pH 7+1M NaCl+1 mM EDTA
The column was initially equilibrated for 7 column volumes of buffer A. After loading of the application, unbound protein was removed by washing using 7 column volumes of buffer A. A linear gradient from 0-100% buffer B for 20 column volumes was used to elute the pACSH239 protease from the column. The fusion protein was eluted from the column at a salt concentration of 0.4-0.5 M NaCl. Some protein was lost in the flow through. However, the pACSH239 protease could be purified to high purity ( ̃85-90%) as determined by HPLC and SDS-PAGE standard methods in a single cation exchange chromatographic step. By LC-MS it was also verified that the eluted pACSH239 protease had a molecular weight as expected.
Purification of HRV14 3C Protease N-Terminally Tagged with SEQ ID NO:10(pACSH238)
Supernatants ready for purification were prepared from cell culture pellets sonicated in 25 mM NaPO4 pH 7, from 80 ml of cell culture as described for pACSH239. A SP Sepharose FF prepacked 5 ml HiTrap column (GE Healthcare) was loaded with the application. The flow rate was 3 ml/min and the buffers used for the purification was:
Buffer A: 50 mM sodium phosphate, pH 7
Buffer B: 50 mM sodium phosphate, pH 7+1M NaCl
The column was initially equilibrated for 7 column volumes with buffer A. Unbound sample was removed in a washing step using 7 column volumes of Buffer A and the pACSH238 protease was eluted from the column using linear gradient from 0-100% buffer B for 20 column volumes.
The pACSH238 protease was eluted from the column at a concentration of  ̃0.2 M NaCl. In contrast to the pACSH239 protease, the major part of the pACSH238 protease bond to the column at these purification conditions. By LC-MS it was also verified that the eluted protease had a molecular weight as expected. The recovery was 4 times higher after the first purification step compared to the pACSH239 protease and the purity was  ̃90% as judged from HPLC and SDS PAGE analysis.
Evaluation of the Activity of SEQ ID NO:19 Tagged HRV14—3C Protease (pACSH239)
The activity of pACSH239 protease was estimated using different amounts of the protease to cleave a fusion protein (pACSH294) comprising the S661 insulin receptor antagonist peptide with a N-terminal tag (SEQ ID NO: 19) and an intervening linker with a modified HRV14 3C cleavage site SSSGGSEVLFQ (SEQ ID NO: 36)
The S661 peptide has the following sequence: GSLDESFYDWFERQLGGGSGGSSLEEEWAQIQCEVWGRGCPSY (SEQ ID NO: 37).
Comparable protein concentrations of the pACSH294 fusion protein substrate and the pACSH239 protease were calculated by integration of UV214 nm absorbance peak areas from analysis of HPLC chromatograms. Enzyme and substrate were mixed in a 200 uL reaction volume and incubated 6 or 12 hours at 25° C. in a cleavage buffer containing 50 mM Tris HCl pH 7.5, 150 mM NaCl using different enzyme to substrate ratios (adjusted according to concentration). LC-MS studies showed that only the S661 peptide (SEQ ID NO: 37) and the released tag (SEQ ID NO: 19)+linker (SEQ ID NO: 36) was observed after 12 hours incubation using 1:100 enzyme to substrate ratio (FIG. 2). Only very small amounts of fusion protein were left after 12 hours using a 1:200 ratio. The results therefore indicate that an enzyme/substrate ratio of 1:100-1:200 is sufficient for complete processing of the pACSH294 fusion protein during a overnight incubation.
The released S661 peptide, present in the digest, could subsequently be purified to more than 98% purity using an anion exchange chromatography column (HiTrap Q sepharose High Performance, 1 ml, GE Healthcare). Buffers and general purification conditions were as described for the purification of the pACSH239 protease. Whereas the S661 peptide bound this column, the pACSH239 protease was not eluted at any salt concentration from the column within the elution gradient, but could only be observed in the flow through as judged by LC-MS analysis of flow through fractions. Thus, the pACSH239 protease was efficiently removed from the target protein in the second purification step.
Evaluation of the Activity of SEQ Id NO:10 Tagged HRV14 3C Protease (pACSH238)
Identical amounts of pACSH238 or pACSH239 protease were used to process the pACSH294 substrate in a buffer consisting of 50 mM Tris HCl pH 7.5, 150 mM NaCl in a parallel experiment. Enzyme to substrate ratios of 1:25 or 1:50 were evaluated for the two enzymes. The amount of S661 insulin receptor antagonist peptide released from the pACSH294 fusion protein after 3 hours incubation at 30° C. was exactly the same for the two enzymes indicating that they have the same specific activity despite different sized tags attached to the N-terminal. As with the pACSH239 protease, the pACSH238 protease did not bind anion exchange column thus indicating that it can be efficiently removed by a suitable purification step following digestion of the target protein precursor.
Example 2
Cloning of Tagged Tobacco Etch Virus Proteases
The mature Tobacco etch virus (TEV) protease has the following amino acid sequence:







GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRN


 


NGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFRE


 


PQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGS


 


PLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRL


 


NNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN (SEQ ID NO: 38)






An insert comprising the TEV sequence with a stabilizing mutation (S219V) was obtained by standard PCR methodology using the Phusion PCR system (Finnzymes), forward and reverse primers comprising XhoI and BamHI cloning site respectively and an existing TEV (Ser219Val) encoding plasmid as template. The 5′-end of the PCR product comprised a sequence encoding a flexible linker (SEQ ID NO: 28).
The PCR product was GFX purified (GE Healthcare) and subcloned in TOPO vectors according the manufacturers (Invitrogen) instructions and as described in Example 1. The correct TEV encoding sequence of XhoI/BamHI inserts from TOPO plasmids was verified by DNA sequencing. Correct inserts were excised from TOPO plasmids with XhoI/BamHI enzymes and fused to the 3′-end of inserts encoding thermostable alkaline tags into pET11a (Novagen) expression vectors. The following constructs were generated:













Product name
Tag
Linker
Processing enzyme







pACSH307
SEQ ID NO: 10
SEQ ID NO: 28
SEQ ID NO: 38


pACSH308
SEQ ID NO: 18
SEQ ID NO: 28
SEQ ID NO: 38


pACSH309
SEQ ID: NO:19
SEQ ID NO: 28
SEQ ID NO: 38









Expression of Tagged TEV Protease Variants.

pACSH307, pACSH308 and pACSH309 protease encoding plasmids were transformed into the Rosetta (DE3)(Novagen) E. coli strains. Transformed cells were probagated on LB (Luria-Bertani) medium agar plates containing 150 micrograms per milliter Ampicillin and 34 micrograms per mL chloramphinicol overnight. Liquid LB medium with ampecillin and chloramphinicol was inoculated from culture plates and cultivated in shaker flasks at 37 degrees C. and 240 rpm. The temperature was lowered to 30° C. when OD600.  ̃0.4-0.5 was reached. After 30 minutes adaption to this temperature, protein induction was initiated by adding 0.5 mM IPTG to the culture medium. The final OD600 obtained after 3 hours induction was in range of 1.6. Cells were harvested by centrifugation. SDS-PAGE of lysates of pellets containing induced and uninduced cells, as well as soluble and insoluble fractions of induced cells (obtained by ultrasonication and centrifugation in a buffer consisting of 10 mM NaPO4 pH 7) was performed to evaluate expression level and solubility of the TEV protease variants. The expression levels were similar for all three constructs. The solubility was estimated to be highest for the SEQ ID NO: 10 tagged TEV variant ( ̃70%) (pACSH307).
Purification of pACSH307 Protease
Purification the pACSH307 protease from sterile filtrated supernatants from culture pellets (from 80 ml culture) sonicated in 25 mM NaPO4 pH 7 was carried out using the AKTA Explorer 100 system (GE Healthcare) with exactly the conditions and buffers described for pACSH238 in Example 1. When separation was performed on HiTrap SP Sepharose FF 5 ml column, a peak was clearly eluted at about 0.25 M NaCl. LC-MS analysis of the fraction from the highest point of the peak gave a molecular mass exactly as expected for this construct and the purity of the main peak was  ̃85-90% as judged by SDS-PAGE and HPLC analysis
Evaluation of the Activity of SEQ ID NO:10 Tagged TEV Protease (pACSH307)
The activity of pACSH307 protease was estimated using different amounts of the protease to cleave a fusion protein (pACSH248) comprising S661 insulin antagonist peptide (SEQ ID NO: 37), as described in example 1 with the same N-terminal tag (SEQ ID NO: 19), but with a linker comprising a Tobacco etch virus cleavage recognition site SSSGGSENLYFQ (SEQ ID NO:39). Digestion experiments were performed in a cleavage buffer containing 50 mM Tris HCl pH 8, 0.5 mM EDTA. LC-MS studies showed that after incubation for 8 hours with an enzyme to substrate ration of 1:1, the major part of the S661 peptide was released from the pACSH248 fusion protein. This verify that the tagged TEV protease is expressed and purified as an active enzyme and can be used to remove the SEQ ID NO:19 tag from the pACSH248 fusion protein.
Example 3
Cloning and Expression of proTGase
The transglutaminase (tGASE) gene consists of a signal peptide and a prodomain followed by the mature tGASE sequence. A protGASE (without signal peptide, but with a N-terminal Met residue) was prepared by PCR from genomic DNA of Streptomyces mobaraensis using standard methodology. The prodomain was altered, so that a HRV14 3C cleavage site was included just before a Gly53-Pro54 N-terminal extension of the mature tGASE sequence. The protGASE sequence was cloned into the pET11a vector (pNNC130) using standard cloning techniques and the protein encoded by this plasmid had the following sequence (the mature tGASE sequence starts with residue 55):







MDNGAGEETKSYAETYRLTADDVANINALNESAPAASSAGPSFRAPLEVLF


 


QGPDSDDRVTPPAEPLDRMPDPYRPSYGRAETVVNNYIRKWQQVYSHRDGR


 


KQQMTEEQREWLSYGCVGVTWVNSGQYPTNRLAFASFDEDRFKNELKNGRP


 


RSGETRAEFEGRVAKESFDEEKGFQRAREVASVMNRALENAHDESAYLDNL


 


KKELANGNDALRNEDARSPFYSALRNTPSFKERNGGNHDPSRMKAVIYSKH


 


FWSGQDRSSSADKRKYGDPDAFRPAPGTGLVDMSRDRNIPRSPTSPGEGFV


 


NFDYGWFGAQTEADADKTVWTHGNHYHAPNGSLGAMHVYESKFRNWSEGYS


 


DFDRGAYVITFIPKSWNTAPDKVKQGWP (SEQ ID NO 40).






The pNNC130 plasmid was transformed into BL21(DE3) (Novagen) and cultivated overnight on LB medium containing 150 micrograms per milliliter Ampecillin. Liquid LB medium containing Ampecillin was inoculated from overnight culture agar plates and cultivated at 37° C. at 240 rpm until OD600 reach  ̃0.8. After 20 min. adaptation to 30° C., expression was induced with 0.1 mM IPTG. The cells were harvested after 4 hours of protein induction at a final OD600 of approximately 2. SDS-PAGE analysis showed that the majority of protGASE was found in the soluble fraction in supernatants obtained after sonication in a buffer containing 10 mM NaPO4 pH 8 and centrifugation of cell debris.
Purification of protGASE:
Cell pellets from 200 ml induced cell culture was dissolved in 40 ml 10 mM NaPO4 buffer pH 8. Following sonication and centrifugation at 10.000 g, supernatants were sterile filtrated and the final application volume was adjusted to 50 ml with 10 mM NaPO4 buffer pH 8.
The purification was performed using the instrumentation described in Example 1 and 2 using a flow rate of 3 ml/min and the following buffers:
Buffer A: 25 mM NaPO4 buffer pH 8.0
Buffer B: 25 mM NaPO4 buffer pH8.0, 0.5M NaCl
A 5 ml Q Sepharose High Performance (HP) HiTrap column (GE Healthcare) was equilibrated for 7 column volumes (CV) with buffer A. The application containing protGASE was loaded on the column and unbound sample was washed out with buffer A for 10 CV. A linear gradient from 0-100% buffer B for 30 column volumes was used to elute the protGASE. A peak containing protGASE was eluted with approximately 0.05 M NaCl concentration from the column. Collected fractions were analyzed by SDS PAGE and the identity of protGASE was confirmed using LC-MS analysis, which gave a mass very close to the expected theoretical mass of the protein
Removal of the Prodomain Using the pACSH238 or pACSH239 Protease
Fractions from the first purification step containing protGASE was pooled and the buffer was changed to a cleavage buffer with 50 mM Tris HCl pH 7.5, 150 mM NaCl and 1 mM DTT using Vivaspin 20 (10000 MWCO) (Satorius) for diafiltration. The amount of purified protGASE and pACSH239 protease was adjusted by estimating UV214 nm absorbance peak areas from HPLC chromatograms. Cleavage tests with different enzyme to substrate ratios (1:10, 1:50, 1:100 and 1:200) were performed with or without 1 mM DTT in the cleavage buffer at 25° C. overnight. Experiments showed that the pACSH239 protease was able to remove the prodomain completely (to obtain a Gly-Pro extended mature tGASE) using an enzyme substrate ration of 1:200 and 1 mM DTT in the cleavage buffer. The mass of the released mature tGASE and the protGASE was determined using LC-MS.
In similar tests, the pACSH238 protease was also found able to efficiently cleave off the prodomain from the protGASE.
Purification of the Mature tGASE:
The Gly-Pro extended mature tGASE obtained after digestion with the pACSH239 protease was diluted to 48 ml in 25 mM NaPO4 pH 6, 1 mM DTT (buffer A) and separated on a 5 ml SP Sepharose High Performance (HP) HiTrap column (GE Healthcare) using the following buffers:
Buffer A: 25 mM NaPO4 buffer pH6.0+1 mM DTT
Buffer B: 25 mM NaPO4 buffer pH6.0+1 mM DTT+0.5M NaCl
Purification was performed using a flow rate of 3 ml/min and linear gradient elution from 0-100% buffer B for 30 column volumes.
UV280 chromatograms from purification indicated that one single major peak was eluted from the SP Sepharose HP column at a NaCl conc. of approx. 0.1 M. LC-MS analysis confirmed the identity of the eluted protein to be mature tGASE. The pACSH239 protease was, due to the high charge of the N-terminally fused SEQ ID NO:19 tag, eluted from the column much later than the mature tGASE and could thus be efficiently removed from the target protein following a second purification step.
Example 4
Synthesis of a HAV18 3C Protease Encoding Sequence Using Overlap PCR
The amino acid sequence of the mature Hepatitis A strain 18 (HAV18) 3C protease is as follows:







STLEIAGLVRKNLVQFGVGEKNGCVRVVVMNALGVKDDWLLVPSHAYKFEK


 


DYEMMEFYFNRGGTYYSISAGNVVIQSLDVGFQDVVLMKVPTIPKFRDITE


 


HFIKKGDVPRALNRLATLVTTVNGTPMLISEGPLKMEEKATYVHKKNDGTT


 


VDLTVDQAWRGKGEGLPGMCGGALVSSNQSIQNAILGIHVAGGNSILVAKL


 


VTQEMFQNIDKKIESQ (SEQ ID NO: 45).






A codon-optimized DNA fragment of mature HAV18 3C protease gene was obtained using splicing by overlap extension PCR methodology with two consecutive steps of PCR as described in example 1 for the HRV14 3C protease. The final fragment with correct sequence contained a 5′-end XhoI and 3′ end BamHI site, which allowed ligation into pET11a vectors (Novagen) so that the SEQ ID NO:10 encoding part was linked to the 5-end of the linker and protease encoding region.













Product name
Tag
Linker
Processing enzyme







pACSH362
SEQ ID NO: 10
SEQ ID NO: 47
SEQ ID NO: 45









Expression of SEQ ID NO: 10 tagged HAV18 3C proteases (pACSH362)

Expression in Rosetta (DE3) E. coli strains was performed essentially as described in example 1. An expression level similar to SEQ ID NO:10 tagged HRV14 3C protease was achievable and at least 80% of the protein was in the soluble fraction after cell disruption and centrifugation.
Purification of SEQ ID NO: 10 taggedHAV18 3C protease (pACSH362)
Cell culture pellets (from 80 ml of culture) of the pACSH362 protease were disrupted by ultrasonication in a buffer containing 25 mM NaPO4 pH 7. Cell debris was spun down by centrifugation at 10.000 g. Supernatants were sterilefiltrated before purification, which was carried out using a AKTA explorer 100 purification system (GE Healthcare). A prepacked SP Sepharose FF HiTrap column with a 5 ml column volume (GE Healthcare) was used for the separation at a flowrate of 3 ml/min using the following buffers:
Buffer A: 50 mM sodium phosphate, pH 7
Buffer B: 50 mM sodium phosphate, pH 7+1M NaCl
The column was initially equilibrated for 7 column volumes of buffer A. After loading of the application, unbound protein was removed by washing using 7 column volumes of buffer A. A linear gradient from 0-100% buffer B for 20 column volumes was used to elute the pACSH362 protease from the column. The pACSH362 protease was eluted from the column at a salt concentration of 0.2-0.3 M NaCl. The major part of the pACSH362 protease bond to the column at these purification conditions. Purity of the eluted pACSH362 protease was estimated to  ̃85-90% as determined by HPLC and SDS-PAGE standard methods after a single cation exchange chromatographic step. By LC-MS it was also verified that the eluted protease had a molecular weight as expected.
Evaluation of the Activity of SEQ ID NO:10 tagged HAV18 3C Protease (pACSH362)
The tagged HAV18 3C protease (pACSH362) was used to process the pACSH360 fusion protein comprising the S661 insulin receptor antagonist peptide (SEQ ID NO: 37) with a N-terminal tag (SEQ ID NO: 19) and an intervening linker with a modified HAV 18 3C cleavage site SSSGGSELRTQ (SEQ ID NO: 49).
A 1:10 or 1:50 enzyme to substrate ratio of the pACSH362 protease and pACSH360 fusion protein was evaluated by LC-MS after incubation for 7 hours at 37° C. A fragment representing the released S661 peptide was observed for both ratios, as in example 1, showing that the tagged HAV18 enzyme is active.
Example 5
Cloning of Tagged Rabbit Hemorrhagic Disease Virus 3C-Like Protease
Purification protein tags from ribosomal protein L9 family coming from Thermus thermophilus, Geobacillus kaustophilus, Thermosipho melanesiensis, BI429 and Acidothermus cellulolyticus 11B were codon optimised for expression E. coli and obtained as synthetic genes from GENEART GmbH, Regensburg, Germany. The gene fragments encoding the tags had an 5′ end NdeI site and a 3′ end XhoI site. A codon optimised fragment with a 5′-end XhoI site and a 3′-end BamHI site encoding the Rabbit Hemorrhagic Disease Virus 3C-like protease (RHDV) was also obtained (GENEART GmbH, Regensburg, Germany).
The following constructs were assembled by means of standard cloning techniques in the pET11a E. coli expression vector in order to generate the following RHDV protease variants:













Product name
Tag
Linker
Processing enzyme







pACSH433
SEQ ID NO: 41
SEQ ID NO: 48
SEQ ID NO: 46


pACSH434
SEQ ID NO: 42
SEQ ID NO: 48
SEQ ID NO: 46


pACSH435
SEQ ID: NO:43
SEQ ID NO: 48
SEQ ID NO: 46


pACSH436
SEQ ID: NO:44
SEQ ID NO: 48
SEQ ID NO: 46









Expression of tagged Rabbit Hemorrhagic Disease Virus 3C-Like Protease

Expression in BL21(DE3) E. coli strains was performed essentially as described in example 1. Very similar expression levels were obtained for all four constructs and the solubility range from about 80%-90% as judged by SDS-PAGE of supernatants and pellets derived after cell disruption and centrifugation.
Purification of SEQ ID NO: 41, 42, 43 and 44 Tagged HAV18 3C Proteases.
Cell culture pellets (from 80 ml of culture) of each of the tagged RHDV proteases were disrupted by ultrasonication. Supernatants were prepared and separated using a prepacked SP Sepharose FF 5 ml column (GE Healthcare) using the same buffers and conditions as described previously.
Upon comparison of recovered amounts of the protease after the capture step, the best result was achieved with the pACSH433 protease comprising the SEQ ID NO: 41 tag, which bound  ̃2-3.5 times more protein on the column than for the remaining variants.
Example 6
Coexpression of Tagged HRV14 3C Protease Variant Together with protGASE (pNNC130) or pACSH294 (S661 Fusion Protein)
The pACYCDuet-1 plasmid (Novagen) has a lower copy number than pET11a (Novagen) plasmid and the amount of protein produced from this plasmid should be lower than the pET11a plasmid. Furthermore, the plasmid contains a different selection marker (chloramphinicol resistance) than pET11a allowing maintenance and co-expression from these two plasmids in the same host strain. It was evaluated whether a method for processing of target protein precursors using tagged processing enzymes was feasible using the pACYCDuet-1 vector for the expression of pACSH238 protease and the pET11a vector for the expression of protGASE (pNNC130) or S661 fusion protein (pACSH294) that can be processed by the pACSH238 protease.
A fragment from the pET11a plasmid encoding pACSH238 protease (Example 1) and a part of the vector backbone upstream of the start codon was excised with MluI and BamHI restriction enzymes and ligated into the MluI/BamHI cleaved pACYCDuet-1 plasmid backbone (Novagen).
The pACYCDuet1 plasmid expressing the pACSH238 protease was transformed into BL21(DE3) together with either the pET11a plasmid encoding the protGASE (pNNC130) or the S661 fusion protein (pACSH294) Thus, one strain can co-express the pACSH238 protease (from the pACYC-Duet-1 plasmid) together with the protGASE and the other strain expresses the pACSH238 protease together with S661 fusion protein (pACSH294).
The co-expressing strains were cultivated in LB medium containing 150 micrograms per milliliter ampicillin and 34 micrograms per milliliter chloramphinicol. in parallel with control strains expressing either the protGASE or the S661 fusion protein alone. Expression conditions were as described for the pACSH238 protease in example 1. After 3 hours expression at 30° C. the co-expressing strains and their controls were harvested. From SDS-PAGE it was apparent that co-expression resulted in a lower molecular weight for the induced protGASE or S661 fusion protein compared to the controls. The truncated induced protein present in the SDS gel band from the pACSH238 protease/protGASE co-expressing strain was identified as tGASE using standard mass spectrometry based methods following in-gel digestion. The results therefore show that the co expressed pACSH238 protease was able to cleave off the prodomain from the protGASE and the SEQ ID NO:19 purification tag from the S661 fusion protein in vivo.

## Claims

1. A processing enzyme comprising an N-terminally attached tag derived from a ribosomal protein from a thermophilic bacteria, wherein at least 15% of the protein's amino acids comprise Lys, Arg or a combination thereof, wherein the tag is attached to the processing enzyme by a linker sequence comprising a sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:33, and SEQ ID NO: 34.

2. The processing enzyme according to claim 1, wherein the tag comprises no cysteine residues.

3. The processing enzyme according to claim 1, wherein the tag comprises from 50 to 250 amino acid residues.

4. The processing enzyme according to claim 1, wherein the tag comprises from 75 to 200 amino acid residues.

5. The processing enzyme according to claim 1, wherein the tag comprises from 100 to 250 amino acid residues.

6. The processing enzyme according to claim 1, wherein the tag comprises from 100 to 200 amino acid residues.

7. The processing enzyme according to claim 1, wherein 15% to 50% of the amino acid residues in the tag comprise Lys, Arg, or a combination thereof.

8. The processing enzyme according to claim 1, wherein 20% to 50% of the amino acid residues in the tag comprise Lys, Arg, or a combination thereof.

9. The processing enzyme according to claim 1, wherein 30% to 50% of the amino acid residues in the tag comprise Lys, Arg, or a combination thereof.

10. The processing enzyme according to claim 1, wherein 40% to 50% of the amino acid residues in the tag comprise Lys, Arg, or a combination thereof.

11. The processing enzyme according to claim 1, wherein 35% to 50% of the amino acid residues in the tag comprise Lys, Arg, or a combination thereof.

12. The processing enzyme according to claim 1, wherein the tag is derived from a large ribosomal subunit protein L9.

13. The processing enzyme according to claim 12, wherein the tag is selected from the group of peptide sequences consisting of SEQ ID NO:10, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

14. The processing enzyme according to claim 1, selected from oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.

15. A method for making a tagged processing enzyme according to claim 1, comprising i) expressing the processing enzyme comprising the N-terminal tag derived from a ribosomal protein from a thermophilic bacteria in a suitable expression host, wherein at least 15% of the protein's amino acids comprise Lys, Arg or a combination thereof, ii) loading the expressed processing enzyme comprising the N-terminal tag on a cation-exchange column, iii) and eluting the processing enzyme comprising the N-terminal tag with a suitable eluent.

16. The method according to claim 15, wherein the processing enzyme is a protease selected from the group consisting of Human Rhino Virus Strain 14 (HRV14) 3C protease, Hepatitis A strain 18 (HAV18) 3C, Tobacco Etch Virus (TEV) protease or Rabbit Hemorrhagic Disease Virus (RHDV) 3C protease.

17. The method according to claim 15, wherein the tag is selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

18. A method for modifying a target protein precursor comprising the steps of contacting the processing enzyme comprising the N-terminal tag according to claim 1 with the target protein precursor and separating the resulting target protein from the processing enzyme.

19. The method according to claim 18, wherein the processing enzyme comprising the N-terminal tag and the target protein precursor are co-expressed in a suitable host enabling isolation of the target protein after expression.