Patent application title: Processing of Peptides and Proteins
Inventors:
Inga Sig Nielsen Norby (Birkerod, DK)
Lars Fogh Iversen (Holte, DK)
Assignees:
NOVO NORDISK A/S
IPC8 Class: AC12N952FI
USPC Class:
435220
Class name: Acting on peptide bond (e.g., thromboplastin, leucine amino-peptidase, etc., (3.4)) proteinase derived from bacteria
Publication date: 2009-02-12
Patent application number: 20090042272
Inventors list |
Agents list |
Assignees list |
List by place |
Classification tree browser |
Top 100 Inventors |
Top 100 Agents |
Top 100 Assignees |
Usenet FAQ Index |
Documents |
Other FAQs |
Patent application title: Processing of Peptides and Proteins
Inventors:
Lars Fogh Iversen
Inga Sig Nielsen Norby
Agents:
NOVO NORDISK, INC.;INTELLECTUAL PROPERTY DEPARTMENT
Assignees:
Novo Nordisk A/S
Origin: PRINCETON, NJ US
IPC8 Class: AC12N952FI
USPC Class:
435220
Abstract:
The invention provides novel methionine aminopeptidase enzymes and their
use.Claims:
1. An E. coli aminopeptidease variant wherein residues Y168, M206, and/or
Q233 are modified such that the variant exhibits extended substrate
specificity in P1' position for Asn, Leu, Ile, Phe, His, Gln, or Trp in
addition to wild type substrate amino acids.
2. The variant of claim 1, wherein the variant includes a modification in position 168.
3. The variant of claim 1, wherein the variant includes a modification in position 206.
4. The variant of claim 1, wherein the variant includes a modification in position 233.
5. The variant of claim 1, wherein the variant includes a modification in positions 206 and 233.
6. The variant of claim 1, wherein the variant includes a modification in positions 168 and 206.
7. The variant of claim 1, wherein the variant includes a modification in positions 168 and 233.
8. The variant of claim 1, wherein the variant includes a modification in positions 168, 206, and 233.
9. The variant of claim 1, wherein position 168 is an Ala.
10. The variant of claim 1, wherein position 206 is an Ala.
11. The variant of claim 1, wherein position 233 is an Ala.
12. The variant of claim 2, wherein position 168 is an Ala.
13. The variant of claim 3, wherein position 168 is an Ala.
14. The variant of claim 4, wherein position 168 is an Ala.
15. An E.coli methionine aminopeptidase having an amino acid sequence selected from Seq. id. No. 3, Seq. id. No. 5, Seq. id. No. 7, and Seq. id. No. 9.
Description:
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001]This patent application is a continuation of copending U.S. patent application Ser. No. 11/424,870, filed Jun. 18, 2006, which is a continuation of International Application PCT/DK2004/000891, filed Dec. 20, 2004, which claims the benefit of U.S. Provisional Patent Application Nos. 60/531,790, filed Dec. 22, 2003 and 60/539,290, filed Jan. 26, 2004 and Danish Patent Application No. PA 2003 01892, filed Dec. 19, 2003.
FIELD OF THE INVENTION
[0002]The invention relates to a method for processing initiator methionine containing proteins by the enzyme Methionine Aminopeptidase and mutants thereof to yield initiator methionine free peptides.
BACKGROUND OF THE INVENTION
[0003]Production of peptides by recombinant techniques using either prokaryotic or eukaryotic expression systems inherently yields the peptide with a leading methionine amino acid. This amino acid may not be present in the native protein i.e. the variant of the peptide processed for translocation. Obtaining the peptide without the leading methionine thus requires a further processing step. In the present invention the step is performed by the enzyme Methionine Aminopeptidase, which selectively cleaves the initiator methionine from the peptide.
[0004]Methionine Aminopeptidases (Met-AP's) are known in the art as enzymes which cleaves leading methionines, if the leading peptide sequence is of a certain predetermined character. Wild-type Escherichia coli Met-AP selectively cleaves after an initiator Met residue if the residue in the P1' position is Gly, Ala, Ser, Thr, Pro, Val or Cys.
[0005]In the present invention the methionine aminopeptidases are improved by introducing mutations in the substrate binding sites which results in methionine aminopeptidases which cleaves the methionine regardless of the leading peptide sequence (P1' position)
SUMMARY OF THE INVENTION
[0006]The invention provides novel mutant methionine aminopeptidases.
[0007]The invention provides isolated DNA encoding such methionine aminopeptidases.
[0008]The invention provides host cells for producing such methionine aminopeptidases.
[0009]The invention provides the use of the mutant methionine aminopeptidase for processing of peptides with an initiator methionine amino acid into a methionine free peptide.
[0010]The invention also provides the processing of specific peptides by mutant methionine aminopeptidases.
[0011]The invention also provides a method for separating the methionine containing starting material from the final cleaved product.
DESCRIPTION OF THE DRAWINGS
[0012]FIG. 1: Example of a E. coli Met-AP mutant expression construct layout. Purification indicates tag for purification purposes. Protease indicates protease cleavage site.
[0013]FIG. 2: Expression in e.g. E. coli of (A) NT1-Enterokinase-Met-AP Y168A or (B) NT1-Enterokinase-Met-AP Y168G, M206N, Q233A as indicated.
[0014]FIG. 3: hexa-His-Xa-Met-AP (M206A, Q233A) cleavage of Met-hIL-21 FIG. 4: Maldi-tof es mass spectrum of purified hexa-His-Xa-Met-AP M206A, Q233A.
[0015]FIG. 5: Purification chromotogram of the separation of the three compounds Met-IL-21, IL-21 and pyroglutamine IL-21.
DEFINITIONS
[0016]P1 defines the first amino acid N-terminal to the recognition site for the enzyme. P1' denotes the amino acid adjacent to P1 towards the C-terminal. P1 in the present invention is methionine.
[0017]In the present invention substrate specificity means selectivity towards the P1' position--which is the position just C-terminal to methionine. Wild-type Escherichia coli Met-AP exhibits the substrate specificity, that it selectively cleaves after an initiator Met residue if the residue in the P1' position is Gly, Ala, Ser, Thr, Pro, Val or Cys. The mutants of the present invention showing an extended substrate specificity means that further aminoacid can occupy the P1' position and still cleavage of the methionine is observed.
[0018]In the context of the present invention variant means a sequence which has maintained the qualitative activity of the parent sequence, ie as methionine aminopeptidase, but wherein the sequence differs from the parent sequence by deletions, insertions, extension or substitution of one or more amino acids of the parent sequence. Variants in principle also includes fragments of any length provided the activity is maintained.
[0019]In the context of the present invention chemical derivatives of a specific protein means a derivative of the native protein which is not a variant, and which maintains the qualitative activity of the parent protein sequence. The chemical derivative includes derivatives such as PEG-groups.
[0020]The terms peptide and proteins are used interchangeable and is not meant as indications or limitations as to size or function of the sequences.
DESCRIPTION OF THE INVENTION
[0021]The Met-Ap from E. coli has a substrate defining pocket (as part of active site) essentially, but probably not exclusively, defined by the amino acids Tyr 168, Met 206 and Gln 233. Mutating these positions extends the enzymes substrate specificity. The novel E.coli aminopeptidases described in the present invention extends the applicability of the Methionine aminopeptidases to be useful for removing the initiating methionine from almost any type of protein or peptide regardless of the amino acid sequence downstream of the methionine (P1' position). Hence, the initiator methionine can be removed from all initiator methionine containing peptides or proteins to produce initiator methionine-free peptides or proteins.
[0022]The E. coli Methionine aminopeptidase gene was cloned and mutant versions have been created using site directed mutagenesis.
[0023]The mutants were expressed in E. coli and the resulting enzymes were purified by conventional His-tag system. The enzyme can also be tagged by for example the FLAG-system or tagged and purified by other technologies as described in WO 03042249. Catalytic activity was monitored using initiator Met containing hIL-21 as a substrate.
[0024]In principle, the invention is generally applicable to any peptide. The invention is demonstrated as being useful for cleavage of the initiator methionine for peptides such as hIL-21. hIL-21 is a model system for P1' position being a Gln. IL-21 is described in WO00/53761 and is described as being effective in the treatment of cancer and viral infection among others. IL-20 is described in WO9927103. hGH refers to human Growth Hormone. Both are model systems for other aminoacids in P1' position.
[0025]In an aspect the invention provides E. coli aminopeptidease variants which are mutated in the active site having extended substrate specificity the P1' position relative to the wild type.
[0026]In an aspect the invention provides the E.coli methionine aminopeptidase variants as described above which extend the substrate specificity in P1' position to include Asn, Leu, Ile, Phe, His, Gln or Trp as well as the aminoacids allowed in position P1' by the wild type.
[0027]In an aspect the invention provides E.coli methionine aminopeptidases as described above, wherein the residues in positions 168, 206 or 233 has been amended into a sequence different from Y168 and/or M206 and/or Q233.
[0028]In an aspect the invention provides E.coli methionine aminopeptidases as described above, comprising amendments of the amino acid in position 168.
[0029]In an aspect the invention provides E.coli methionine aminopeptidases as described above comprising amendment in position 206.
[0030]In an aspect the invention provides E.coli methionine aminopeptidases as described above comprising amendment in position 233.
[0031]In an aspect the invention provides E.coli methionine aminopeptidases as described above, comprising amendments in position 206 and 233.
[0032]In an aspect the invention provides E.coli methionine aminopeptidases as described above comprising amendments in position 168 and 206.
[0033]In an aspect the invention provides E.coli methionine aminopeptidases as described above comprising amendments in position 168 and 233.
[0034]In an aspect the invention provides E.coli methionine aminopeptidase as described above, comprising amendments in positions 168, 206 and 233.
[0035]In an aspect the invention provides E.coli methionine aminopeptidases as described above, wherein the amendments comprises exchange of wildtype amino acid into Gly, Ala, Ser, Thr, Asn or Asp.
[0036]In an aspect the invention provides E.coli methionine aminopeptidases as described above, wherein the amendments comprises Ala and/or Gly.
[0037]In an aspect the invention provides E.coli methionine aminopeptidases as described above, wherein the amendments comprises Ala.
[0038]In an aspect the invention provides E.coli methionine aminopeptidase as described above, wherein position 168 is Ala.
[0039]In an aspect the invention provides E.coli methionine aminopeptidase as described above, wherein position 206 is Ala.
[0040]In an aspect the invention provides. E.coli methionine aminopeptidase as described above, wherein position 233 is Ala.
[0041]The invention thus provides the methionine aminopeptidase enzyme having the following sequence (also described as seq. id. no. 1)
MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHG YPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRI TQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXa CGHGIGRGFHEEPQVLH YDSRETNVVLKPGMTFTIEPXb VNAG KKEIRTMKDGWTVKTKDRSLSAXx YEHTIVVT DNGCEILTLRKDDTIPAIISHDE,
[0042]wherein Xa, Xb and Xc are variable amino acids, and wherein Xa, Xb and Xc are not simultaneously Tyr, Met and Gln respectively. In an aspect of the invention one or more of Xa, Xb and Xc are exchanged from the wild type amino acid into Gly, Ala, Ser, Thr, Asn or Asp. In an aspect of the invention Xa, Xb and Xc are exchanged from the wild type amino acid into Gly or Ala;. In an aspect of the invention Xa, Xb and Xc are exchanged from the wild type amino acid into Ala.
The present invention thus provides substitution Y168 to Ala (Y168A)(Seq. id no. 9) MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYH- G YPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRI TQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREACGHGIGRGFHEEPQVLHYDSRETN VVLKPGMTFFIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDT IPAIISHDEand the corresponding DNA encoding the above as seq. id. no. 8;
[0043]The present invention thus provides substitution Met 206 to Ala (M206A)(Seq. id no. 3)
[0044]MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSA CLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMG ERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRGFHEEPQVLHY DSRETNVVLKPGMTFTFIEPAVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILT LRKDDTIPAIISHDE,
which extends the enzymes substrate specificity to allow the following amino acids: Asn, Leu, Ile and Phe in the P1' position.The present invention also provides substituting Gln 233 to Ala (Q233A) (Seq. id. No.5 )MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSA CLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMG ERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRGFHEEPQVLHY DSRETNVVLKPGMTFFIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAAYEHTIVVTDNGCEILT LRKDDTIPAIISHDE,
[0045]or both Met 206 and Gln 233 into Ala (M206A Q233A)(Seq. id no. 7):
[0046]MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSA CLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMG ERLCRITQESLYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREXCGHGIGRGFHEEPQVLHY DSRETNVVLKPGMTFFIEPAVNAGKKEIRTMKDGWTVKTKDRSLSAAYEHTIVVTDNGCEILT LRKDDTIPAIISHDE,
which further allow the P1' amino acids to be His, Gln and Trp.
[0047]In aspects of the invention postion 168 is amended into Gly (Y168G) or Ala (Y168A) or Asn (Y168N). Aspects of the invention are wherein amino acid 206 is an Ala (M206A) or a Gly (M206G) or Asn (M206N), and/or wherein amino acid 233 is an Ala (Q233A) or a Gly (Q233G) or Asn (Q233N). Aspects of the invention comprise the combination of two or three amendments according to the below,--wherein the wild-type combination of (Y168 M206 233Q) is not within the invention.
TABLE-US-00001 Position 168 Position 206 Position 233 A/G/N/Y A/G/N/M A/G/N/Q
Accordingly, aspects of the invention are wherein position 206 and position 233 are both Ala (M206A Q233A) or Gly or Asn, or combinations thereof: (M206G Q233A), (M206G Q233G), (M206A Q233G), (M206N Q233A), (M206N Q233N), (M206A Q233N). Aspects of the invention are wherein postion 168 is amended according to the below:
TABLE-US-00002 Position 168 Position 206 Position 233 A/G/N A Q A/G/N G Q A/G/N N Q A/G/N M A A/G/N M G A/G/N M N A/G/N A A A/G/N G A A/G/N N A A/G/N A G A/G/N A N A/G/N N G A/G/N N N A/G/N G G A/G/N G N
Aspects of the invention are wherein at least one of the amended positions are amended into an Ala.Aspects of the invention are the following mutants: (Y168G M206A), (Y168G M206A 233A), (Y168G M206N), (Y168G M206N 233A), (Y168A M206A 233A), (Y168A M206A), (Y168A M206N), (Y168A M206N 233A) and (M206A Q233A);
[0048]The invention thus provides a novel enzymes capable of cleaving a peptide containing an initiating methionine followed by a Asn, Leu, Ile, Phe, His, Gln or Trp in the P1' postion as well as the amino acids allowed by the wild type E.coli aminopeptidase. Wildtype E.coli methionine aminopeptidase allows the P1' to be any of the following amino acids: Gly, Ala, Ser, Thr, Pro, Val or Cys.
The invention thus also provides recombinant DNA molecules encoding the sequence above. The DNA sequences are disclosed in Seq. id. no. 2, 4 and 6.The invention also provides specifically the DNA encoding the sequences for the mutants M206A, Q233A or M206A Q233A) above.In the present invention the mutant Methionine aminopeptidases are expressed in E. coli, but in principle the host cells could be of other prokaryotic origin or eukaryotic origin such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris etc. or for example mammalian cells.The invention thus provides host cells transformed by the recombinant DNA molecule of above.Removal of initiator methionine by methionine aminopeptidase may be performed in vitro following methionine aminopeptidase expression in, and purification from, prokaryotic or eukaryotic cells. This procedure is demonstrated below. Alternatively removal of initiator methionine may take place in vivo either in cells expressing a di-cistronic plasmid or in cells co-expressing plasmids carrying the methionine aminopeptidase and the substrate peptide or protein. In vivo initiator methionine processing may also be performed in cells where the genes encoding the methionine aminopeptidase and the peptide or protein to be processed have been integrated into the genome.
[0049]Experiments have been performed which provides a set of optimum conditions for the reaction: The optimal temperature for the reaction was determined to be between 15 and 24 degrees Celsius. Typically the reaction was hereafter performed at 18 degrees Celsius.
[0050]The concentration of ZnCl2 was determined to be optimal at around 7.5 μM and NaCl concentration was found optimal around 100 mM and acceptable under 130 mM.
[0051]After cleavage of the initiator methionine separation of the product from the starting material can be achieved by exploiting the different biophysical properties of the two peptides.
In an embodiment of the invention the peptide is hIL-21, which after removal of the initial methionine contains a Gln in the N-terminal. Treatment with Qcyclase forms a pyroglutamine (pGlu). Due to cyclic amid formation the products net change is negative relative to the Methionine containing peptide. The difference in charge affects the eluation on a cation exchange column, due to Methionine containing peptide having a stronger binding to the cation resin. Further, in non-cyclised hIL-21 the N-terminal positioned Gln residue will have the ability to form a hydrogen bond between the side chain amide oxygen and the charged N-terminal backbone amine and thereby masking the charge at the N-terminus. Met-hIL-21 will not possess the ability for a similar charge masking and will therefore bind stronger to the cation exchange column than hIL-21.
[0052]In an embodiment of the invention, a method seperation of protein mixtures between identical proteins starting with Met-Gln and Gln respectively is provided.
[0053]In a specific embodiment of the invention seperation of Met-hIL-21 and hIL-21 is provided.
[0054]In another specific embodiment of the invention separation of Met-hIL-21 and hIL-21 and mutants thereof is provided.
EXAMPLES
[0055]Various Met-AP expression constructs, as outlined in FIG. 1, have been created. NT1 (HHHNSWDHDINR) or hexa-His tag has been added to the various mutant forms of Met-AP for purification purposes. The purification tag may be removed using Factor Xa in some constructs or Enterokinase in others, or the purification tag may be left on the enzyme. mRNA expression was under the control of the T7 or the tac promoter. Constructs under the control of the T7 promoter were expressed in BL21(DE3) whereas constructs under the control of the tac promoter were expressed in BL21. Expression was induced by addition of IPTG to 0.4 mM to cultures (6 mL) grown to OD600 0.4 in LB-medium. Cells were harvested by centrifugation after 2.5 hours. Cell lysis was done by multiple freeze-thaw cycles and soluble or insoluble protein fractions were separated by centrifugation. Soluble or insoluble protein, before or after induction of expression, originating from equal amounts cells (measured by OD600) were subjected to SDS-PAGE and subsequent colloidal blue staining (FIG. 2). Met-AP expression levels were estimated at ˜250 mg/L after 2.5 h of induction in 6 mL cultures.
[0056]E. coli cells harvested from 1 L of culture expressing hexa-His-Met-AP M206A, Q233A were lysed using a cell disruptor, and the clarified lysate was applied on a Ni2+-NTA superflow column. Elution with an imidazole gradient released the Met-AP fusion protein at approximately 200 mM imidazole. The enzyme was further purified and buffer exchanged (into storage/cleavage buffer) using size exclusion chromatography. The enzyme was analysed using SDS-PAGE, MALDI-MS and N-terminus sequencing--verifying the molecular mass and identity of the enzyme.
[0057]According to the procedure above NT1-Enterokinase-Met-AP mutants were prepared. Expression was under the control of the tac promoter. Addition of IPTG to the cultures induced primarily soluble expression of the Met-AP enzymes. The following mutants were prepared according to the above: (Y168G M206A), (Y168G M206A 233A), (Y168G M206N), (Y168G M206N 233A), (Y168A M206A 233A), (Y168A M206A), (Y168A M206N) and (Y168A M206N 233A)
[0058]Hexa-His-Xa-MetAP Q233A was affinity purified using Ni2+-NTA superflow. Maldi-tof es mass spectrum of purified hexa-His-Xa-Met-AP M206A, Q233A shows that the correct enzymes were isolated. A mass of 32038.90 corresponds to Met-hexa-His-Xa-Met-AP M206A, Q233A and a mass of 31942.10 corresponds to hexa-His-Xa-Met-AP M206A, Q233A indicating that hexa-His-Xa-Met-AP M206A. Q233A was processed by WT Met-AP or hexa-His-Xa-Met-AP M206A, Q233A in vivo. The result is demonstrated in FIG. 4.
[0059]Addition of hexa-His-Xa-Met-AP M206A, Q233A to Met-hIL-21 at pH 7, 18° C. generated ˜65% Met-free hIL-21. In 44 h. more than 90% cleavage of Met-hIL-21 could be observed (FIG. 3).
[0060]Another mutant prepared by this method was hexa-His-Xa-Met-AP Q233A;
Removal of Initiator Met from Met-IL21 by Met-AP (M206A, Q233A).
[0061]Purified Met-AP (M206A, Q233A) was used to remove the initiator Methionine from partly or fully purified Met-IL21. The cleavage was performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 mM NaCl. 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8. The cleavage was assayed by MALDI-TOF spectroscopy. The time of reaction was 2-66 hours. Using these condition removal of Methionine from Met-IL21 below detection limits of Met-IL21 could be performed.
Removal of Initiator Met from Met-IL21 by Met-AP (M206A).
[0062]Purified Met-AP (M206A) was used to remove the initiator Methionine from partly or fully purified Met-IL21. The cleavage was performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 mM NaCl, 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8. The cleavage was assayed by MALDI-TOF spectroscopy. The time of reaction was 2-66 hours. Using these condition removal of Methionine from Met-IL21 below detection limits of Met-IL21 could be performed.
Influence of Temperature on Removal of Initiator Met from Met-IL21 by Met-AP (M206A, Q233A).
[0063]Using the conditions and assay described in example 1 the temperature was varied between 4, 15, 24 and 30 degrees Celsius, respectively while the other parameters was fixed. The optimal temperature for the reaction was determined to be between 15 and 24 degrees Celsius. Typically the reaction was hereafter performed at 18 degrees Celsius.
Influence of ZnCl2 Concentration on Removal of Initiator Met from Met-IL21 by Met-AP (M206A, Q233A).
[0064]Using the conditions and assay described in example 1 the ZnCl2 concentration was varied between 7.5, 11 and 15 μM, respectively while the other parameters were fixed. The optimal ZnCl2 concentration for the reaction was determined to be 7.5 μM. Typically, the reaction was hereafter performed at 7.5 μM ZnCl2.
Influence of NaCl Concentration on Removal of initiator Met from Met-IL21 by Met-AP (M206A, Q233A).
[0065]Using the conditions and assay described in example 1 the NaCl concentration was varied between 80, 130 and 180 mM, respectively while the other parameters were fixed. The maximum NaCl concentration tolerated for the reaction to run was determined to be 130 mM. Typically the reaction was hereafter performed at 100 mM NaCl.
Influence of the Addition of Q-cyclase on Removal of Initiator Met from Met-IL21 by Met-AP (M206A, Q233A) and the Formation of Pyro-Glutamine.
[0066]Using the conditions as described in examples above the effect of adding Q-cyclase to the reaction mixture was determined. Again MALDI-TOF was used for assaying the removal of Methonine and subsequently conversion of glutamine in position 1 in IL21 into pyro-glutamine. It was found that the addition of Q-cyclase to the reaction mixture did not negatively influence the removal of initiator Methionine from Met-IL21 and further the Q-cyclase was fully efficient in converting converting glutamine in position 1 in IL21 into pyro-glutamine under the reaction conditions described in the examples above.
Purification and Separation of Met-IL21, IL21 and Pyro-Glutamine IL-21 using a Mono-S Column.
[0067]The different bio-physical properties between Met-IL21, IL21 and pyro-glutamine IL21 can be used for purification purposes/separation. Pyro-glutamine IL21 will due to the cyclized amid formation lack the normal protonation of the N-terminus. The (-1) charge difference between hIl-21 starting with pyro-glutamine and Met-IL21 can be used on a cation exchange column that will elute pyro-glutamine IL21 first (due to its lack of one positive charge) and subsequently Met-IL21 which displays a stronger binding to the cation resin. Further, in non-cyclized IL21 the N-terminally positioned glutamine will have the ability to form a hydrogen bond between the side chain amide oxygen and the charged N-terminal backbone amine, and thereby masking the charge at the N-terminus. Met-IL21 will not poses the ability for a similar charge masking and will therefore bind stronger to a cation exchange column than IL21. A mixture of Met-IL21, IL21 and pyro-glytamine IL21 including 300 mM NaCl and buffered at pH 6.5 was loaded on a Mono-S column. The A buffer consisted of 300 mM NaCl buffered at pH 6.5 and the B buffer 1 M NaCl buffered at pH 6.5. A linear gradient (performed on an AKTA system) from 0-20% B buffer was applied over 45 column volumes. The fractions was assayed as described under the Q-cyclase example above. Using the above described gradient, efficient separation of Met-IL21, IL21. pyro-glutamine IL21 was achieved.(FIG. 5)
Met-hGH
[0068]Purified Met-AP (M206A) is used to remove the initiator Methionine from partly or fully purified Met-hGH (human growth hormone) where the P1' is a Phe residue. The cleavage is performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 mM NaCl, 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8. The cleavage is assayed by MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Using these conditions partly or full removal of Methionine from Met-hGH is demonstrated.
Met-hGH
[0069]Purified Met-AP (M206A, Q233A) is used to remove the initiator Methionine from partly or fully purified Met-hGH (human growth hormone) where the P1' is a Phe residue. The cleavage is performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 mM NaCl, 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8. The cleavage is assayed by MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Using these conditions partly or full removal of Methionine from Met-hGH is achieved.
Met-IL-20
[0070]Purified Met-AP (M206A) is used to remove the initiator Methionine from partly or fully purified Met-IL-20 where the P1' is a Leu residue. The cleavage is performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 mM NaCl, 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8. The cleavage is assayed by MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Using these conditions partly or full removal of Methionine from Met-IL-20 is demonstrated.
Met-IL-20
[0071]Purified Met-AP (M206A, Q233A) is used to remove the initiator Methionine from partly or fully purified Met-IL-20 where the P1' is a Leu residue. The cleavage is performed in a reaction buffer typically consisting of the following components: 2-100 mM K2SO4, 2-500 MNaCl, 1-100 μM ZnCl2 and 2-30 mM Hepes buffer pH 6-8.
[0072]The cleavage is assayed by MALDI-TOF spectroscopy. The time of reaction is 2-66 hours. Using these conditions partly or full removal of Methionine from Met-IL-20 is demonstrated.
Sequence CWU
1
91264PRTEscherichia colimisc_feature(168)..(168)Xaa can be any naturally
occurring amino acid 1Met Ala Ile Ser Ile Lys Thr Pro Glu Asp Ile Glu Lys
Met Arg Val1 5 10 15Ala
Gly Arg Leu Ala Ala Glu Val Leu Glu Met Ile Glu Pro Tyr Val20
25 30Lys Pro Gly Val Ser Thr Gly Glu Leu Asp Arg
Ile Cys Asn Asp Tyr35 40 45Ile Val Asn
Glu Gln His Ala Val Ser Ala Cys Leu Gly Tyr His Gly50 55
60Tyr Pro Lys Ser Val Cys Ile Ser Ile Asn Glu Val Val
Cys His Gly65 70 75
80Ile Pro Asp Asp Ala Lys Leu Leu Lys Asp Gly Asp Ile Val Asn Ile85
90 95Asp Val Thr Val Ile Lys Asp Gly Phe His
Gly Asp Thr Ser Lys Met100 105 110Phe Ile
Val Gly Lys Pro Thr Ile Met Gly Glu Arg Leu Cys Arg Ile115
120 125Thr Gln Glu Ser Leu Tyr Leu Ala Leu Arg Met Val
Lys Pro Gly Ile130 135 140Asn Leu Arg Glu
Ile Gly Ala Ala Ile Gln Lys Phe Val Glu Ala Glu145 150
155 160Gly Phe Ser Val Val Arg Glu Xaa Cys
Gly His Gly Ile Gly Arg Gly165 170 175Phe
His Glu Glu Pro Gln Val Leu His Tyr Asp Ser Arg Glu Thr Asn180
185 190Val Val Leu Lys Pro Gly Met Thr Phe Thr Ile
Glu Pro Xaa Val Asn195 200 205Ala Gly Lys
Lys Glu Ile Arg Thr Met Lys Asp Gly Trp Thr Val Lys210
215 220Thr Lys Asp Arg Ser Leu Ser Ala Xaa Tyr Glu His
Thr Ile Val Val225 230 235
240Thr Asp Asn Gly Cys Glu Ile Leu Thr Leu Arg Lys Asp Asp Thr Ile245
250 255Pro Ala Ile Ile Ser His Asp
Glu2602792DNAEscherichia coli 2atggctatct caatcaagac cccagaagat
atcgaaaaaa tgcgcgtcgc tggccgactg 60gctgccgaag tgctggagat gatcgaaccg
tatgttaaac cgggcgtcag caccggcgag 120ctggatcgca tctgtaatga ttacattgtt
aatgaacaac acgcggtttc tgcctgcctc 180ggctatcacg gctatccgaa atccgtttgc
atctctatta atgaagtggt gtgccacggt 240atcccggacg atgctaagct gctgaaagat
ggcgatatcg ttaacattga tgtcaccgta 300atcaaagatg gtttccacgg cgatacctcg
aaaatgttta tcgtcggtaa gccgaccatc 360atgggcgaac gtctgtgccg catcacgcaa
gaaagcctgt acctggcgct acgcatggta 420aaaccaggca ttaatctgcg cgaaatcggt
gcggcgattc agaaatttgt cgaagcagaa 480ggcttctccg tcgttcgtga atattgcgga
cacggtattg gtcgcggctt ccatgaagaa 540ccgcaggtgc tgcactatga ctcccgtgaa
accaacgtcg tactgaaacc tgggatgacg 600ttcaccatcg agccagcggt caacgcgggt
aaaaaagaga tccgcaccat gaaagatggc 660tggacggtaa aaaccaaaga tcgcagcttg
tctgcacagt atgagcatac tattgtggtg 720actgataacg gctgcgaaat tctgacgcta
cgcaaggatg acaccatccc ggcgataatc 780tcgcacgacg aa
7923264PRTEscherichia coli 3Met Ala Ile
Ser Ile Lys Thr Pro Glu Asp Ile Glu Lys Met Arg Val1 5
10 15Ala Gly Arg Leu Ala Ala Glu Val Leu
Glu Met Ile Glu Pro Tyr Val20 25 30Lys
Pro Gly Val Ser Thr Gly Glu Leu Asp Arg Ile Cys Asn Asp Tyr35
40 45Ile Val Asn Glu Gln His Ala Val Ser Ala Cys
Leu Gly Tyr His Gly50 55 60Tyr Pro Lys
Ser Val Cys Ile Ser Ile Asn Glu Val Val Cys His Gly65 70
75 80Ile Pro Asp Asp Ala Lys Leu Leu
Lys Asp Gly Asp Ile Val Asn Ile85 90
95Asp Val Thr Val Ile Lys Asp Gly Phe His Gly Asp Thr Ser Lys Met100
105 110Phe Ile Val Gly Lys Pro Thr Ile Met Gly
Glu Arg Leu Cys Arg Ile115 120 125Thr Gln
Glu Ser Leu Tyr Leu Ala Leu Arg Met Val Lys Pro Gly Ile130
135 140Asn Leu Arg Glu Ile Gly Ala Ala Ile Gln Lys Phe
Val Glu Ala Glu145 150 155
160Gly Phe Ser Val Val Arg Glu Tyr Cys Gly His Gly Ile Gly Arg Gly165
170 175Phe His Glu Glu Pro Gln Val Leu His
Tyr Asp Ser Arg Glu Thr Asn180 185 190Val
Val Leu Lys Pro Gly Met Thr Phe Thr Ile Glu Pro Ala Val Asn195
200 205Ala Gly Lys Lys Glu Ile Arg Thr Met Lys Asp
Gly Trp Thr Val Lys210 215 220Thr Lys Asp
Arg Ser Leu Ser Ala Gln Tyr Glu His Thr Ile Val Val225
230 235 240Thr Asp Asn Gly Cys Glu Ile
Leu Thr Leu Arg Lys Asp Asp Thr Ile245 250
255Pro Ala Ile Ile Ser His Asp Glu2604792DNAEscherichia coli 4atggctatct
caatcaagac cccagaagat atcgaaaaaa tgcgcgtcgc tggccgactg 60gctgccgaag
tgctggagat gatcgaaccg tatgttaaac cgggcgtcag caccggcgag 120ctggatcgca
tctgtaatga ttacattgtt aatgaacaac acgcggtttc tgcctgcctc 180ggctatcacg
gctatccgaa atccgtttgc atctctatta atgaagtggt gtgccacggt 240atcccggacg
atgctaagct gctgaaagat ggcgatatcg ttaacattga tgtcaccgta 300atcaaagatg
gtttccacgg cgatacctcg aaaatgttta tcgtcggtaa gccgaccatc 360atgggcgaac
gtctgtgccg catcacgcaa gaaagcctgt acctggcgct acgcatggta 420aaaccaggca
ttaatctgcg cgaaatcggt gcggcgattc agaaatttgt cgaagcagaa 480ggcttctccg
tcgttcgtga atattgcgga cacggtattg gtcgcggctt ccatgaagaa 540ccgcaggtgc
tgcactatga ctcccgtgaa accaacgtcg tactgaaacc tgggatgacg 600ttcaccatcg
agccaatggt caacgcgggt aaaaaagaga tccgcaccat gaaagatggc 660tggacggtaa
aaaccaaaga tcgcagcttg tctgcagcgt atgagcatac tattgtggtg 720actgataacg
gctgcgaaat tctgacgcta cgcaaggatg acaccatccc ggcgataatc 780tcgcacgacg
aa
7925264PRTEscherichia coli 5Met Ala Ile Ser Ile Lys Thr Pro Glu Asp Ile
Glu Lys Met Arg Val1 5 10
15Ala Gly Arg Leu Ala Ala Glu Val Leu Glu Met Ile Glu Pro Tyr Val20
25 30Lys Pro Gly Val Ser Thr Gly Glu Leu Asp
Arg Ile Cys Asn Asp Tyr35 40 45Ile Val
Asn Glu Gln His Ala Val Ser Ala Cys Leu Gly Tyr His Gly50
55 60Tyr Pro Lys Ser Val Cys Ile Ser Ile Asn Glu Val
Val Cys His Gly65 70 75
80Ile Pro Asp Asp Ala Lys Leu Leu Lys Asp Gly Asp Ile Val Asn Ile85
90 95Asp Val Thr Val Ile Lys Asp Gly Phe His
Gly Asp Thr Ser Lys Met100 105 110Phe Ile
Val Gly Lys Pro Thr Ile Met Gly Glu Arg Leu Cys Arg Ile115
120 125Thr Gln Glu Ser Leu Tyr Leu Ala Leu Arg Met Val
Lys Pro Gly Ile130 135 140Asn Leu Arg Glu
Ile Gly Ala Ala Ile Gln Lys Phe Val Glu Ala Glu145 150
155 160Gly Phe Ser Val Val Arg Glu Tyr Cys
Gly His Gly Ile Gly Arg Gly165 170 175Phe
His Glu Glu Pro Gln Val Leu His Tyr Asp Ser Arg Glu Thr Asn180
185 190Val Val Leu Lys Pro Gly Met Thr Phe Thr Ile
Glu Pro Met Val Asn195 200 205Ala Gly Lys
Lys Glu Ile Arg Thr Met Lys Asp Gly Trp Thr Val Lys210
215 220Thr Lys Asp Arg Ser Leu Ser Ala Ala Tyr Glu His
Thr Ile Val Val225 230 235
240Thr Asp Asn Gly Cys Glu Ile Leu Thr Leu Arg Lys Asp Asp Thr Ile245
250 255Pro Ala Ile Ile Ser His Asp
Glu2606792DNAEscherichia coli 6atggctatct caatcaagac cccagaagat
atcgaaaaaa tgcgcgtcgc tggccgactg 60gctgccgaag tgctggagat gatcgaaccg
tatgttaaac cgggcgtcag caccggcgag 120ctggatcgca tctgtaatga ttacattgtt
aatgaacaac acgcggtttc tgcctgcctc 180ggctatcacg gctatccgaa atccgtttgc
atctctatta atgaagtggt gtgccacggt 240atcccggacg atgctaagct gctgaaagat
ggcgatatcg ttaacattga tgtcaccgta 300atcaaagatg gtttccacgg cgatacctcg
aaaatgttta tcgtcggtaa gccgaccatc 360atgggcgaac gtctgtgccg catcacgcaa
gaaagcctgt acctggcgct acgcatggta 420aaaccaggca ttaatctgcg cgaaatcggt
gcggcgattc agaaatttgt cgaagcagaa 480ggcttctccg tcgttcgtga atattgcgga
cacggtattg gtcgcggctt ccatgaagaa 540ccgcaggtgc tgcactatga ctcccgtgaa
accaacgtcg tactgaaacc tgggatgacg 600ttcaccatcg agccagcggt caacgcgggt
aaaaaagaga tccgcaccat gaaagatggc 660tggacggtaa aaaccaaaga tcgcagcttg
tctgcagcgt atgagcatac tattgtggtg 720actgataacg gctgcgaaat tctgacgcta
cgcaaggatg acaccatccc ggcgataatc 780tcgcacgacg aa
7927264PRTEscherichia coli 7Met Ala Ile
Ser Ile Lys Thr Pro Glu Asp Ile Glu Lys Met Arg Val1 5
10 15Ala Gly Arg Leu Ala Ala Glu Val Leu
Glu Met Ile Glu Pro Tyr Val20 25 30Lys
Pro Gly Val Ser Thr Gly Glu Leu Asp Arg Ile Cys Asn Asp Tyr35
40 45Ile Val Asn Glu Gln His Ala Val Ser Ala Cys
Leu Gly Tyr His Gly50 55 60Tyr Pro Lys
Ser Val Cys Ile Ser Ile Asn Glu Val Val Cys His Gly65 70
75 80Ile Pro Asp Asp Ala Lys Leu Leu
Lys Asp Gly Asp Ile Val Asn Ile85 90
95Asp Val Thr Val Ile Lys Asp Gly Phe His Gly Asp Thr Ser Lys Met100
105 110Phe Ile Val Gly Lys Pro Thr Ile Met Gly
Glu Arg Leu Cys Arg Ile115 120 125Thr Gln
Glu Ser Leu Tyr Leu Ala Leu Arg Met Val Lys Pro Gly Ile130
135 140Asn Leu Arg Glu Ile Gly Ala Ala Ile Gln Lys Phe
Val Glu Ala Glu145 150 155
160Gly Phe Ser Val Val Arg Glu Tyr Cys Gly His Gly Ile Gly Arg Gly165
170 175Phe His Glu Glu Pro Gln Val Leu His
Tyr Asp Ser Arg Glu Thr Asn180 185 190Val
Val Leu Lys Pro Gly Met Thr Phe Thr Ile Glu Pro Ala Val Asn195
200 205Ala Gly Lys Lys Glu Ile Arg Thr Met Lys Asp
Gly Trp Thr Val Lys210 215 220Thr Lys Asp
Arg Ser Leu Ser Ala Ala Tyr Glu His Thr Ile Val Val225
230 235 240Thr Asp Asn Gly Cys Glu Ile
Leu Thr Leu Arg Lys Asp Asp Thr Ile245 250
255Pro Ala Ile Ile Ser His Asp Glu2608795DNAEscherichia coli 8atggctatct
caatcaagac cccagaagat atcgaaaaaa tgcgcgtcgc tggccgactg 60gctgccgaag
tgctggagat gatcgaaccg tatgttaaac cgggcgtcag caccggcgag 120ctggatcgca
tctgtaatga ttacattgtt aatgaacaac acgcggtttc tgcctgcctc 180ggctatcacg
gctatccgaa atccgtttgc atctctatta atgaagtggt gtgccacggt 240atcccggacg
atgctaagct gctgaaagat ggcgatatcg ttaacattga tgtcaccgta 300atcaaagatg
gtttccacgg cgatacctcg aaaatgttta tcgtcggtaa gccgaccatc 360atgggcgaac
gtctgtgccg catcacgcaa gaaagcctgt acctggcgct acgcatggta 420aaaccaggca
ttaatctgcg cgaaatcggt gcggcgattc agaaatttgt cgaagcagaa 480ggcttctccg
tcgttcgtga agcgtgcgga cacggtattg gtcgcggctt ccatgaagaa 540ccgcaggtgc
tgcactatga ctcccgtgaa accaacgtcg tactgaaacc tgggatgacg 600ttcaccatcg
agccaatggt caacgcgggt aaaaaagaga tccgcaccat gaaagatggc 660tggacggtaa
aaaccaaaga tcgcagcttg tctgcacaat atgagcatac tattgtggtg 720actgataacg
gctgcgaaat tctgacgcta cgcaaggatg acaccatccc ggcgataatc 780tcgcacgacg
aataa
7959264PRTEscherichia coli 9Met Ala Ile Ser Ile Lys Thr Pro Glu Asp Ile
Glu Lys Met Arg Val1 5 10
15Ala Gly Arg Leu Ala Ala Glu Val Leu Glu Met Ile Glu Pro Tyr Val20
25 30Lys Pro Gly Val Ser Thr Gly Glu Leu Asp
Arg Ile Cys Asn Asp Tyr35 40 45Ile Val
Asn Glu Gln His Ala Val Ser Ala Cys Leu Gly Tyr His Gly50
55 60Tyr Pro Lys Ser Val Cys Ile Ser Ile Asn Glu Val
Val Cys His Gly65 70 75
80Ile Pro Asp Asp Ala Lys Leu Leu Lys Asp Gly Asp Ile Val Asn Ile85
90 95Asp Val Thr Val Ile Lys Asp Gly Phe His
Gly Asp Thr Ser Lys Met100 105 110Phe Ile
Val Gly Lys Pro Thr Ile Met Gly Glu Arg Leu Cys Arg Ile115
120 125Thr Gln Glu Ser Leu Tyr Leu Ala Leu Arg Met Val
Lys Pro Gly Ile130 135 140Asn Leu Arg Glu
Ile Gly Ala Ala Ile Gln Lys Phe Val Glu Ala Glu145 150
155 160Gly Phe Ser Val Val Arg Glu Ala Cys
Gly His Gly Ile Gly Arg Gly165 170 175Phe
His Glu Glu Pro Gln Val Leu His Tyr Asp Ser Arg Glu Thr Asn180
185 190Val Val Leu Lys Pro Gly Met Thr Phe Thr Ile
Glu Pro Met Val Asn195 200 205Ala Gly Lys
Lys Glu Ile Arg Thr Met Lys Asp Gly Trp Thr Val Lys210
215 220Thr Lys Asp Arg Ser Leu Ser Ala Gln Tyr Glu His
Thr Ile Val Val225 230 235
240Thr Asp Asn Gly Cys Glu Ile Leu Thr Leu Arg Lys Asp Asp Thr Ile245
250 255Pro Ala Ile Ile Ser His Asp Glu260
User Contributions:
comments("1"); ?> comment_form("1"); ?>Inventors list |
Agents list |
Assignees list |
List by place |
Classification tree browser |
Top 100 Inventors |
Top 100 Agents |
Top 100 Assignees |
Usenet FAQ Index |
Documents |
Other FAQs |
User Contributions:
Comment about this patent or add new information about this topic: