Stable recombinant adenosine deaminase
SUBSTANCE: isolated recombinant adenosine deaminase is described, which comprises polypeptide SEQ ID NO: 1 or a version polypeptide SEQ ID NO: 1 of isolated recombinant adenosine deaminase, where the version polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions selected from the group consisting of: Gin instead Lysl98; Ala instead Thr245; and Arg instead of Gly351, and DNA encoding it. The conjugate of polyalkylene oxide with the said adenosine deaminase for treatment of adenosine deaminase-mediated diseases is presented where adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa for ADA protein. The methods of purification of the recombinant adenosine deaminase are proposed, including protein purification using ion exchange chromatography, or protein purification using hydrophobic interaction chromatography. Also the preparations of recombinant adenosine deaminase produced by these methods are provided.
EFFECT: invention enables to obtain recombinant adenosine deaminase, having increased stability.
14 cl, 1 tbl, 10 ex
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the priority of provisional patent application U.S. 60/913,009, filed April 20, 2007, the contents of which are incorporated herein by reference.
The technical FIELD TO WHICH the INVENTION RELATES.
The invention provides recombinant adelaideans, mutated to increase stability.
The LEVEL of TECHNOLOGY
Adelaideans (ADA) used in the treatment of diseases caused by enzyme deficiency called severe combined immunodeficiency (DID) or "bubble-boy" (boy in the bubble), for some time. More than 15 years, the company Enzon pharmaceuticals did therapeutic HELL available to patients in the form Paglierani HELL, obtained using the bovine enzyme HELL.
Recently, attempts were made to replace the bovine enzyme recombinant enzyme (hereinafter "the Council"). As a substitute of purified natural bullish HELL was viewed as recombinant human ("Rada"), and recombinant bovine ("rbada") enzymes. Enzymes rbada and rcad somewhat less stable than the native purified bovine enzyme, which is used currently. And rcad, and rbada believed to decompose the mechanism, matched with the decomposition of the cyst is on: adding oxygen; formation developed; the acceleration of decomposition with increasing pH; deposition, especially at elevated pH and concentration of samples. In restored condition cysteine contains a reactive SH group (sulfhydryl), and in this form is responsible for the decay.
Experimental data suggest that the only external cysteine may be responsible for the degradation observed for rbada, and for Rada. The structure of bovine ADA (i.e., native bullish HELL, purified from bovine source) is substantially similar to the structure rcade: bullish and HELL, and rcad have the same number cysteines in the same positions of the primary sequence. Receive recombinant human and recombinant bovine ADA contain degradation products/impurities (developed)that are consistent with the reactivity of cysteine. Native bovine ADA differs in structure from recombinant bovine HELL the fact that native bovine HELL with each mol of HELL has one mol of cysteine. In addition, native bullish HELL is stable at high pH, suggesting that cysteine is associated with HELL, functions as a blocking group.
One method of stabilization of recombinant human and/or recombinant bovine HELL is kierowanie active mod is and Cys (Cys 74 in Mature rbada and Mature rcad) any of oxidized glutathione, iodoacetamide, iodixanol acid, cystine, other developed and mixtures thereof. This method is described in application for U.S. patent 11/738,012 (the present applicant), entitled "Stabilized proteins" (Stabilized Proteins), the contents of which are by reference incorporated herein in full.
Notwithstanding the foregoing, the elimination of the need for additional stages of kupirovaniya by changing the protein structure to ensure its own stability directly if the expression is preferred. In U.S. patent 5346823 described stabilization prokaryotic proteases, such as subtilisin, and neutral proteases by replacing destabilizing residues Cys to Ser and other amino acid residues, through mutations. However, mutational analysis of the active sites in the HELL have shown that substitution of Cys residue (Cys 262) led to a significant reduction of enzyme activity, Bhaumik et al 1993, The J. of Biol. Chem., 268 (8):5464-5470. Thus, until the present invention, the stabilization of adenoidectomies by replacing the active and external Cys residue with other amino acid residue with maintaining optimal useful activity of the enzyme was not known.
Thus, it is advantageous to provide sustainable rbada and rcade, that is, without significant decomposition during storage and processing, while the pH levels used in the op is emalina the Pegylation of the enzyme.
Thus, the invention provides recombinant HELL, in which any oxidizable cysteine residue replaced with a non-oxidizing amino acid residue compared to the form of the wild-type enzyme HELL. Mutein HELL includes a non-oxidizing acid balance, which is one of the natural L-amino acids, e.g. alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, Proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine and/or known in the prior art variants and derivatives of the natural L-amino acids, for example, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidino acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoethanol acid, 3-aminoadamantane acid, 2-aminopimelic acid, 2,4-diaminoalkanes acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n-ethylasparagine, hydroxylysine, ALLO-hydroxylysine, 3-hydroxyprolin, 4-hydroxyprolin, isodesmosine, ALLO-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-meth what Lisina, n-methylvaline, Norvaline, norleucine, ornithine and the like, Optional, methionine or tryptophan are excluded because they are potentially oxidizable.
More preferably, a non-oxidizing amino acid residue is one of the serine, alanine, asparagine, glutamine, glycine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine. Serine is the most preferred. In some preferred embodiments, the implementation of the oxidizable cysteine is approximately 74 the position of the Mature protein of HELL. Recombinant ADA preferably is recombinant bovine ADA or recombinant HELL man, that is, for example, translated from the DNA molecule corresponding to SEQ ID NO: 2 or SEQ ID NO: 4, and which preferably consists of SEQ ID NO: 1 or SEQ ID NO: 3. When recombinant HELL is recombinant bovine ADA corresponding to SEQ ID NO: 1, ADA optional is expressed with a polymorphism selected from one or more Gln instead of Lys198; Ala instead of Thr245; Arg instead Gly351.
The invention also provides a conjugate polyalkyloxy-HELL, where polyalkylene preferably is polyethylene glycol. Optional polyethylene glycol anywhereman with recombinant adenozindeaminaza through a chemical linker selected from the group consisting of succinonitrile is the thiazolidinedione, urethane, succinimidylester and linkers based on amides. It is preferable to succinimidylester. The polyethylene glycol preferably covalently attached to the Epsilon-amino group of Lys recombinant adelaideans.
Conjugate peg-ADA includes at least 1 (i.e. one or more) polietilenglikolya chains attached to the Epsilon-amino groups, preferably at least 5 (five or more) polietilenglikolya chains attached to the Epsilon-amino groups, or, more preferably, from about 11 to about 18 polietilenglikolya chains attached to the Epsilon-amino groups of residues Lys recombinant HELL.
The polyethylene glycol conjugates of the invention has a molecular weight of from about 2000 to about 100000 kDa, or, more preferably, from about 4000 to about 45000 kDa.
The invention additionally provides a method of purification of recombinant adelaideans invention. For example, recombinant adelaideans preferably purified using ion exchange chromatography (for example, Capto Q, DEAE and SP chromatography), and recombinant adelaideans SEQ ID NO: 1 is preferably purified using hydrophobic interaction chromatography.
In addition, the present invented the e additionally provides a method for the treatment of ADA-mediated diseases in mammals, including the introduction of an effective amount of recombinant HELL according to the present invention. HELL-mediated disease includes, for example, TKID, cancer, etc.
DETAILED description of the INVENTION
The present invention provides a stable recombinant enzymes - adelaideans. Adelaideans inventions get by replacing the cysteine residue which is subjected to the oxidation process, when the enzyme is in solution, an acceptable alternative amino acid residue that remains active, charge and tertiary structure of the enzyme while eliminating the source of the destructive instability.
In order to ensure clarity in the description of the invention below are definitions of several terms.
The term "recombinant" refers to a protein obtained using cells, which in their native state do not contain endogenous copies of DNA that can Express this protein. Cells produce recombinant protein, because they have been genetically altered through the introduction of appropriate selected nucleic acid sequence. The term also includes a reference to a cell, or nucleic acid, or vector, which has been modified through the introduction of a heterologous (exogenous or alien) well Lanovoy acid or by modifying the native nucleic acid with the receipt of the form, which is not native to the cell, or the specified cell obtained from cells modified in this way. Thus, for example, recombinant cells Express genes that are not present in native (non-recombinant) form of the cells, Express a mutant genes, which are present in native form or Express native genes that are otherwise improperly expressed, expressed insufficiently or not expressed at all.
Used in the present description "nucleic acid" or "sequence of nucleic acids" include a reference to deoxyribonucleotide or ribonucleotidic polymer or in single or double form, and if there are no limits encompass known analogs of natural nucleotides that hybridize to nucleic acids is similar to the natural nucleotides. Unless otherwise indicated, a particular sequence of nucleic acids includes corresponding complementary sequence.
The term "encoding" in relation to a specified nucleic acid includes a reference to nucleic acids, which include information for broadcast in a particular protein. Information is determined by codons.
"A host cell" is a cell that can support replication or e is cpressey expression vector. Cell host may be prokaryotic cells such as E. coli cells, or eukaryotic, such as yeast cells, insect amfibie or mammals.
Used in this description, "polypeptide", "peptide" and "protein" are used interchangeably and include a link to a polymer composed of amino acid residues.
The term "residue" or "amino acid residue"or "amino acid" includes reference to an amino acid, which is part of the protein, polypeptide or peptide (collectively, the "peptide").
The amino acid may be a natural amino acid and, if there are no limits, can encompass known analogs of natural amino acids that can function similarly to natural amino acids.
"Transfection" refers to the absorption of the expression vector of the host-cell, regardless of whether expressed any coding sequences are in fact, or not. Numerous methods of transfection are known to the average person skilled in the art. For example, the transfection is carried out in the presence of the expression vector and high concentrations CaPO4using electroporation, using a phage or viral expression vector for installation in a cell of the host, by means of mechanical embedding nucleic acids, and even by culturing cells-Ho who Aina in the presence of loose fragments of nucleic acids. Transfection is usually considered successful when the cell host there is any indication of the target vector.
"Transformation" refers to the introduction of nucleic acids into the body so that the nucleic acid is capable of replication either as an extrachromosomal element or by integration into the host chromosome. Depending on the host cell transformation performed by applying known from the prior art methods, specific cells-owners. Calcium treatment using calcium chloride as described by Cohen, S. N. Proc. Natl. Acad. Sci. (USA), 69:2110 (1972) and Mandel et al, J. Mol. Biol. 53:154 (1970), usually used for prokaryotic or other cells that have a cell wall (for example, many bacterial and/or plant cells). For mammalian cells without such cell walls, the preferred method of precipitation of calcium phosphate Graham, F. and van der Eb, A., Virology, 52: 456-457 (1978). General aspects of system transformation master of mammalian cells have been described in U.S. patent 4,399,216, published on 16 August 1983. Transformation of yeast are typically carried out according to the method of Van Solingen, P., et a!., J. Bad., 130: 946 (1977) and Hsiao, C. L., et al., Proc. Natl. Acad. Sci, (USA) 76: 3829 (1979). But it can also use any other known from the prior art methods of introducing nekleenov the th acid, for example, DNA, cells, such as, for example, microinjection, lipofection or fusion of protoplasts.
Used in the present description, the term "complementary" as applied to nucleic acid refers to the steam room opposite chain synthesized (using the principle of complementarity Watson-Crick) replication of the first nucleic acid molecule using a given molecule as a matrix for the formation of a new, second chain nucleic acids. In one aspect of the invention two molecules of nucleic acid are complementary to each other, when they hybridize or bind under stringent conditions.
The term "functionally linked" refers to such adjacent locations, for example, the regulatory region and an open reading frame, which may be the normal functioning of the elements. Thus, the open reading frame, which is functionally linked with regulatory sequences refers to a configuration in which the coding sequence can be expressed under the control of these two sequences.
"Regulatory sequences" refers to sequences of nucleic acids that are required for the expression of the operatively linked coding sequence in the specification is Eskom the body of the host. Regulatory sequences that are suitable for prokaryotes, for example, include a promoter, optionally a sequence operator, the binding site of the ribosome and possibly other as yet poorly understood sequence. Eukaryotic cells are known to use, for example, such regulatory sequences as promoters, polyadenylation signals, and enhancers, as well as many others.
"Expression system" or "expression vector" refer to sequences of the nucleic acid containing the target coding sequence and regulatory sequences in functional relation by which organisms are the hosts, transformed these sequences are capable of producing the encoded proteins. To implement the transformation of the expression system may be included in the vector, however, then the corresponding nucleic acid molecule can also be integrated into the chromosome of the host body.
Used in this description, "cell", "cell line" and "cell culture" are used interchangeably, with all such designations include progeny. Thus, "transformants" or "transformed cells" include the primary target cell and cultures derived from it without the number of passages. You should also understand the ü, that all progeny may not be strictly identical in genomic content, due to deliberate or accidental mutations. Also included mutant offspring with the same functional properties, which are detected in the originally transformed cell. When necessary other definitions, it is clear from the context.
In the framework of the present invention, the term "residue", as should be understood, means that part of the connection to which it refers, for example, PEG, HELL, amino acid, etc. that remains after it has undergone a substitution reaction with another compound.
In the framework of the present invention the term "polymer residue" for example, "the rest PEG", as should be understood, means that part of a polymer or PEG, which remains after it has undergone a reaction with other compounds, groups, etc.
In the framework of the present invention, the term "alkyl"used in the present description, refers to a saturated aliphatic hydrocarbon, including linear, branched and cyclic alkyl groups. The term "alkyl" includes alkyl-thio-alkyl, alkoxyalkyl, cycloalkylation, heterocytolysine and C1-6-alkylcarboxylic group. Preferably, the alkyl group contains 1-12 carbon atoms. More preferably, lilina group is lower alkyl, comprising from about 1-7 carbon atoms, most preferably about 1-4 carbon atoms. The alkyl group may be substituted or unsubstituted. When an alkyl group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilane, cycloalkyl, cycloalkenyl, heteroseksualci, heteroaryl, alkenyl, quinil, C1-6-hydrocarbonyl, aryl, and amino.
In the framework of the present invention the term "substituted"used in the present description, refers to adding or replacing one or more atoms contained within a functional group or join one of the groups selected from halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilane, cycloalkyl, cycloalkenyl, heteroseksualci, heteroaryl, alkenyl, quinil, C1-6-carbonyl, aryl, and amino.
The term "alkenyl"used in the present description, refers to groups containing at least one carbon-carbon double bond, including linear, branched and cyclic groups. Preferably, Alchemilla group includes priblizitelen the 2-12 carbon atoms. More preferably, Alchemilla group is the lowest alkenyl comprising from about 2-7 carbon atoms, most preferably about 2-4 carbon atoms. Alchemilla group can be substituted or unsubstituted. When Alchemilla group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, cyano, nitro, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilane, cycloalkyl, cycloalkenyl, heteroseksualci, heteroaryl, alkenyl, quinil, C1-6-alkylcarboxylic, aryl, and amino.
The term "quinil"used in the present description, refers to groups containing at least one carbon-carbon triple bond, including linear, branched and cyclic groups. Preferably Alchemilla group includes approximately 2-12 carbon atoms. More preferably Alchemilla group is the lowest quinil comprising from about 2-7 carbon atoms, most preferably about 2-4 carbon atoms. Alchemilla group can be substituted or unsubstituted. When Alchemilla group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, regulatel, hydroxyl, mercapto, hydroxy, cyano, alkylsilane, cycloalkyl, cycloalkenyl, heteroseksualci, heteroaryl, alkenyl, quinil, C1-6-hydrocarbonyl, aryl, and amino. Examples of "quinil include propargyl, propyne and 3-hexyne.
In the framework of the present invention, the term "aryl" refers to aromatic hydrocarbon ring system containing at least one aromatic ring. Aromatic ring optionally may be condensed or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalen and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.
In the framework of the present invention, the term "cycloalkyl" refers to cyclic C3-8the hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. cycloheptyl and cyclooctyl.
In the framework of the present invention, the term "cycloalkenyl" refers to cyclic C3-8the hydrocarbon containing at least one carbon-carbon double bond. Examples cycloalkenyl include cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,3-cyclohexadienyl, cycloheptenyl, cycloheptatriene and cyclooctanol.
Under this the image is placed, the term "cycloalkenyl" refers to an alkyl group, substituted C3-8-cycloalkyl group. Examples cycloalkylation group include cyclopropylmethyl and cyclopentolate.
In the framework of the present invention, the term "alkoxy" refers to an alkyl group with the specified number of carbon atoms attached to the molecule through an oxygen bridge. Examples of alkoxygroup include, for example, methoxy, ethoxy, propoxy, isopropoxy.
In the framework of the present invention "alcylaryl" group refers to an aryl group, a substituted alkyl group.
In the framework of the present invention "kalkilya" group refers to an alkyl group, substituted aryl group.
In the framework of the present invention, the term "alkoxyalkyl" refers to an alkyl group substituted by alkoxygroup.
In the framework of the present invention, the term "alkyl-thio-alkyl" refers to alkyl-S-alkyl thioester group, such as methylthio methyl or methylthioethyl.
In the framework of the present invention, the term "amino" refers to a nitrogen-containing group, which is known from the prior art, derived from ammonia by replacing one or more hydrogen radicals by organic radicals. For example, the terms "acylamino and alkylamino" refer to certain organic radicals, N-substituted aryl and alkyl replacement groups, with the NGOs.
In the framework of the present invention, the term "alkylaryl" refers to a carbonyl group, a substituted alkyl group.
In the framework of the present invention, the terms "halogen" or "halo" refer to fluorine, chlorine, bromine and iodine.
In the framework of the present invention, the term "heteroseksualci" refers to non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen and sulfur. Geteroseksualnoe ring optionally may be condensed or otherwise attached to other heteroseksualnymi rings and/or non-aromatic hydrocarbon rings. Preferred heterocytolysine groups include from 3 to 7 members. Examples geterotsiklicheskikh groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine and pyrazole. Preferred heterocytolysine group include piperidinyl, piperazinil, morpholinyl and pyrrolidinyl.
In the framework of the present invention, the term "heteroaryl" refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen and sulfur. Heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocyclyl the ilen rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothiazyl, pyridyl, hinely, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolin, benzisothiazole, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl and benzimidazolyl.
In the framework of the present invention, the term "heteroatom" refers to nitrogen, oxygen and sulphur.
In some embodiments, the implementation substituted alkali include carboxyacid, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethyl; replaced alkenyl include carboxyaldehyde, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethanol; substituted alkinyl include carboxykinase, aminoalkyl, dialkylamino, hydroxyalkyl and mercaptoethanol; substituted cycloalkyl include groups such as 4-chlorocyclohexane; arily include groups such as naphthyl; substituted arily include groups such as 3-bromophenyl; aralkyl include groups such as tolyl; heteroalkyl include such groups as ethylthiophene; substituted heteroalkyl include groups such as 3-methoxy-thiophene; alkoxy includes groups such as methoxy; and phenoxy includes such g is uppy, as 3-nitrophenoxy. "Halo", as should be understood, includes fluorine, chlorine, iodine and bromine.
In the framework of the present invention "positive integer", as should be understood, includes an integer greater than or equal to 1 and, as will be clear to the average specialists in this area, is within the scope of rationality.
In the framework of the present invention, the term "associated"as should be understood, includes covalent (preferred) or non-covalent joining of one group to another, that is, the result of a chemical reaction.
The concept of "effective amount" and "sufficient" within the framework of the present invention shall mean the amount that achieves the desired effect or therapeutic effect, and the effect as such is understandable to the average experts in this field.
In the framework of the present invention, the term "adenosine", as should be understood, includes the nucleosides adenosine and deoxyadenosine. Adenosine also includes adenosine and deoxyadenosine present in the form of AMP, ADP, ATP, damp, DADF or dATP.
In the framework of the present invention "adenosine-mediated disease" or "adelaideans-reactive disease", as should be understood, broadly includes any disease, pathological condition, or impairment, which has successfully used the introduction And The a or its active fraction, and so, regardless of the route of administration.
In the framework of the present invention "treatment of adenosine-mediated disease" or "treatment adelaideans-reactive diseases, such as DID, as should be understood, means eliminating, minimizing or reducing the symptoms or conditions, compared with that observed in the absence of treatment with the use of HELL. The degree of recovery can be confirmed, for example, by reducing the level of adenosine.
In a broad sense, the treatment of adenosine-mediated disease should be considered successful when the desired clinical response. In the alternative, the success of treatment can confirm, having at least 20%, or preferably 30%, more preferably 40%or higher (i.e., 50%or 80%) reduction in the level of adenosine, including other clinical indicators, considered an expert in this area, compared to that observed in the absence of treatment with the use of HELL.
In addition, the use of terms in the singular for convenience of description in no way be considered limiting in any way. Thus, for example, reference to a composition including an enzyme, refers to one or more molecules of this enzyme. It should also be understood that the present invention is not limited to specific configurations stages of methods and materials, described in this application, since such configuration, stage methods and materials may vary slightly.
It should also be understood that the terminology used in this application, is used only for the purpose of describing particular embodiments and should not be limiting, since the scope of the present invention will be determined by the appended claims and its equivalents.
B. Enzymes HELL, obtained by genetic engineering methods
Initial attempts to obtain recombinant enzyme ADA, including enzymes expressed from human or bovine genes revealed instability during storage, not observed previously for natural HELL, obtained from the intestinal tract of cattle. Studies degradation products Rada and rbada confirmed that both enzymes HELL decay mechanism, matched with the decomposition of cysteine. For example, adding oxygen to rcad leads to the formation of more hydrophilic compounds than rcade with a mass of 16 and 32 Da higher than that rcade. In addition, it leads to the formation of developed (as indicated by the reversion of the formation of degradation products in the add dithiothreitol [DTT]); increasing the intensity of decomposition with increasing pH; deposition, especially at higher pH and at high con is entrale samples suggesting the possibility of the formation of intermolecular disulfide bonds, leading to the formation of insoluble aggregates.
The authors of the present invention have determined that the only external cysteine is responsible for the degradation observed for Rada. Bullish (non-biodegradable) HELL has a structure similar to the structure rcade: bullish and HELL, and rcad contain the same number of cysteines in the same positions of the primary sequence. This rbada also contains degradation products/impurities (developed)that are consistent with the reactivity of cysteine. Native bovine ADA differs in structure from rbada the fact that one mole of cysteine associated with each mol of HELL, and native bovine ADA stable at high pH, suggesting that the cysteine associated with HELL, functions as a blocking group. Cysteine associated with native bovine ADA may be removed by treatment with a reducing agent, such as monotypical or DTT. Not wanting to associate it with any theory or hypothesis, it can be assumed that the cysteine group is associated with HELL by disulfide bonds, as follows:
where one cysteine in the primary sequence HELL associated with a molecule of cysteine. Cysteine present in such disulfide linkages are resistant to oxidative degradation processes, mentioned in the first section. The remains of the cysteine located at positions 74, 152, 153, 168 and 261 human and bovine Mature HELL. The study of three-dimensional structure of bovine ADA, obtained using x-ray analysis (Kinoshita et al., 2005, Biochemistry, 44:10562-10569) showed that cysteine at positions 74, 152, 153, 168 and 261 may not participate in the formation of intramolecular disulfide bonds. Structural geometric constraints, as is known, in General, prevent the formation of disulfide bonds vicinal cysteine residues, such as those that are in positions 152 and 153 of HELL. Thus, all cysteine residues are potentially in a reduced state and, therefore, are potential candidate sites for reactions of oxidative degradation. However, the above visual analysis of the three-dimensional structure of bovine ADA showed that cysteine 74 definitely exposed to solvent in a greater degree than the other four cysteines and, moreover, that the other four cysteine, apparently, are absorbed into the structure of the enzyme to the extent that a significant interaction with solvated reactants, probably, prevented (if the protein is not are denatured). The existence of a single reactive cysteine residue could explain managerialization native who ICLEI adelaideans, that, apparently, is the result of posttranslational modification.
The facts presented above indicate that reactive cysteine at position 74 may be responsible for the degradation observed for rcad and rbad, and that kierowanie reactive-S-H group of the cysteine protects rcade or rbada obvious from oxidative degradation observed for these recombinant enzymes. To confirm that conducted the following experiment. Recombinant offspring at a concentration of approximately 0.6 mg/ml was treated with 125 mm iodoacetamide (IAA) in sodium phosphate buffer with a pH of 7.4 for 16 hours at 37°C. for several minutes after start of the reaction, the analysis of samples using RP-HPLC with UV and mass spectrometric detection showed that approximately 70.9 per cent rcade were managerialization IAA, and 17.2 per cent were subjected to derivatization at two sites. After incubation for 2 and 16 hours chromatographic profile was not significantly changed, which indicates that the derivative was resistant to oxidative degradation, typical rcade. Then prepared like sample rcade, but without IAA, which was analyzed in the same way. After incubation for 16 hours at 37°C and pH of 7.4 protein Rada degraded by 30% (degree of decomposition of the sample in comparison with the similar degradation). The results correspond to a single prevailing external cysteine, which can be protected by kupirovaniya iodoacetamide. These experiments are described in more detail in U.S. patent (the present applicant) 11/738,012, entitled "Stabilized proteins included in the present description by reference as specified above.
Although kierowanie effectively prevents oxidative degradation of reactive cysteine in HELL, the application of such blocked enzyme requires additional process steps. Thus, it was investigated the direct elimination of unstable Cys residue of the encoding gene by substituting another amino acid. Suitable for replacing amino acid is an amino acid that is not subject to the same type of oxidation, it does not impair the formation of the tertiary structure of the folded protein HELL, and in the usual form of the invention chosen so as not to be subjected to random binding with activated polyalkyleneglycol in the process of forming the conjugate. Suitable for replacement of oxidizable cysteine according to the invention is any of the known in the prior art of natural amino acids and/or unnatural amino acids and/or their derivatives that meet the specified criteria. A sample list of such amino acids including the em natural L-amino acids, such as: alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, Proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine. Tryptophan and methionine can be oxidized relatively easily and in some optional embodiments of implementation are less preferred.
Methods of production of recombinant proteins using site-specific incorporation of unnatural amino acids into cells-the hosts described in the literature, for example, Liu et al., 2007, Nat. Methods 4(3):239-44, Xie et al., 2006 Nat. Rev. Mol. Cell. Biol. 7(10):775-82, Ryu et al., 2006, Nat. Methods 3(4):263-65, Deiters et al, 2004, Bioorg. Med. Chem Lett. 14(23):5743-5, Bogosian et al., 1989, J. Biol. Chem. 264(1):531-9, Tang et al., 2002, Biochemistry 41(34):10635-45, Budisa et al., 1995. Eur. J. Biochem. 230(2): 788-96 and Randhawa et al., 1994, Biochemistry, 33(14):4352-62. Thus, substituting amino acid may also include modified or less typical amino acid, such as 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidine acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoethanol acid, 3-aminoadamantane acid, 2-aminopimelic acid, 2,4-diaminopentane acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethylglycine, n-Atlasa the OO ahin, hydroxylysine, ALLO-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, ALLO-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n-methylvaline, Norvaline, norleucine and ornithine.
Preferred natural amino acids, which are optionally substituted cysteine in recombinant ADA include, for example, alanine, serine, aspargine, glutamine, glycine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine. Serine is the most preferred and cited as an example in the future.
Thus, were received expressing human and bovine adelaideans wild-type DNA molecules subjected to optimize codons for expression in E. coli and mutation to expression mutein rbada and muteena rcade, including the Ser residue at position 74 of the respective Mature proteins (position 75 translated protein) instead of natural Cys residue. This Ser74-rbada (SEQ ID NO: 1) and Ser74-rcad (SEQ ID NO: 3), respectively. In addition, it should be noted that natural bullish HELL isolated from intestinal tract of cattle, also contains 6 residues, excision removed from the C-terminal region. An optional characteristic of the present invention is that Ser74-rbada according to the invention is expressed or without 6 C-terminal residues (in the form of moutai is a), or is subjected to post-translational modifications in order to remove these 6 C-terminal residues that are absent in the purified natural bullish HELL.
It should also be noted that natural bullish HELL isolated from intestinal tract of cattle, has polymorphisms in relation to SEQ ID NO: 5, polymorphisms of the prey of HELL include, for example, glutamine at position 198 instead of lysine, alanine at position 245 instead of threonine; arginine at position 351 instead of glycine. Thus, it is expected that recombinant mutein (position 74) bullish HELL according to the invention may also include additional substitutions in one or more of those provisions, or analogues of these provisions: Gln instead of Lys198; Ala instead of Thr245; Arg instead Gly351.
In an additional aspect of the invention the present invention provides a selected DNA that encode mutein HELL, having the amino acid sequence according to SEQ ID NO:1 or SEQ ID NO:3 described in this application. Other DNA encoding mutein HELL with one or more substitutions: Gln instead of Lys198; Ala instead of Thr245; Arg instead Gly351also included in the scope of the present invention.
Suitable expression vector may be derived from genomic or cDNA that encodes rcade or rbada, respectively, which are not necessarily under to what stralem suitable functionally linked inducible promoter. DNA is preferably codon-optimized for the appropriate host cell and mutated using any suitable known from the prior art method, for example, high-performance siteprovides mutagenesis using oligonucleotides (Olsen DB and Eckstein F, Proc Natl Acad Sci USA 87:1451-5; 1990), synthesis of full length genes using overlapping long oligonucleotides (N Vasantha and Filpula D., Gene 76:53-60; 3989), PCR-mediated gene synthesis (K Jayaraman et al., Proc Natl Acad Sci USA 88:4084-88: 1991) or PCR extension overlapping primers (Pogulis RJ et al., Methods Mol Biol 57:167-76; 1996).
Typically, prokaryotes are preferred for the initial cloning of DNA sequences and vector constructs used in the invention. For example, the most preferred is a strain of E. coli K12 294 MM (ATCC No.31,446). Other microbial strains which may be used just as an example, include strains of E. coli, as E. coli B and E. coli X1776 (ATCC No.31,537). Can be used the aforementioned strains, as well as, for example, E. coli W3110 (F-, lambda-, prototrophic, ATCC No.27,325), K5772 (ATCC No.53,635) and SR101, bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various species of Pseudomonas.
Typically, plasmid vectors containing replicon and regulatory sequences derived from a species compatible sklejkoj-master, used together with the above organisms-owners. Conventional plasmid vectors represent denitive DNA molecules, preferably containing the recognition sites of the enzymes that are suitable for integration of exogenous DNA sequences, selective gene for resistance to the antibiotic, the site of initiation of replication for Autonomous reproduction in the cell is the master gene for selection or selection of clones containing an insert of the recombinant DNA. Available plasmid vectors suitable for use in E. coli cells, include, for example, pET3, pET9, pET11, and other series of pET (included in the catalogue of the Corporation Novagen), pBAD, trc, phoA, trp and plasmids OL/R/PL/R.
Just as an example, E. coli is typically transformed using plasmid pBR322 derived from E. coli (see, e.g., Bolivar et al., 1977, Gene. 2:95). pBR322 contains genes for resistance to ampicillin and tetracycline, and provides, therefore, a simple method for the identification of transformed cells. Similarly, the plasmid pUC are a convenient vectors for selection and replication of DNA molecules (Yanisch-Perron, et al., 1985, Gene 33: 103-119, the contents of which are fully incorporated into the present application by reference). Plasmid pBR322 or other microbial plasmid or phage must also contain, or modified to include promoters that can be used by the microbial organism is La expression of its own encoded proteins.
Promoters commonly used in recombinant DNA construction include the system on the basis of promoter beta-lactamase (penitsillinazy) and lactose promoter (Chang et al., 1978, Nature, 375:615; Itakura ef al., 1977, Science, 198:1056; Goeddel et al., 1979, Nature, 281:544), and tryptophan (trp) promoter (Goeddel et al., 1980, Nucleic Acids Res., 8:4057; EPO Appl. Publ. No.0036,776). They are used most often, but have been opened and used, other microbial promoters, published nucleotide sequence that allows qualified functionally integrate them into known vectors, for example, plasmid vectors.
Just as an example, regulation of transcription in E. coli can be provided by any of the following inducible promoters include lac, trp, phoA, araBAD, T7, trc and derivatives lambda promoters PLand PRas well as others known in the prior art (e.g., Makrides, 1996, Environ. Rev. 60:512-538, the contents of which are fully incorporated into the present application by reference).
Suitable conditions of induction is not necessarily compatible with the vector include, for example, adding arabinose, lactose or thermal induction, lack of phosphates, lack of tryptophan and so on. Preferably, iuzirosadym element is the Lac-operon that is induced by isopropylthioxanthone (IPTG).
A suitable signal sequence (signal peptide) can the be obtained from pelB, fd pIII or ompA.
Suitable selective markers of resistance to antibiotics known in the art and include, for example, those that give resistance to ampicillin, kanamycin, chloramphenicol, rifampicin and tetracycline, among others.
The appropriate sequence of initiation of replication include the corresponding sequences of the following plasmids: pUC19, pACYC117, pUB110, pE194, pAMB1, pIJ702, pBR322, pBR327 and pSC101.
Suitable termination sequences include, for example, the main terminator of phage fd, TΦ and rrnB.
In addition to prokaryotes can also be used eukaryotic microorganisms such as yeast culture. From eukaryotic microorganisms most commonly used Saccharomyces cerevisiae, or common Baker's yeast, although there are many other available strains. For expression in Saccharomyces commonly used plasmid YRp7, for example, Stinchcomb et al., 1979, Nature, 282:39; Kingsman et al., 1979, Gene, 7:141; Tschemper et al., 1980, Gene, 10:157). This plasmid already contains the trp1 gene, which provides a selective marker of choice for a mutant strain of yeast lacking the ability to grow in the absence of tryptophan, for example ATCC No.44,076 or PEP4-1 (Jones, 1977, Genetics, 85:12). The presence of abnormalities in trp1, which is the feature of the genome of the yeast host cell, provides an effective medium for the detection of transformants grew up on the y in the absence of tryptophan.
The expression system based on Pichia pastoris, as shown, provides a high level of production of several proteins (Cregg, J.M. et al., 1993, Bio/Technology 11:905-910, the contents of which are fully incorporated into the present description by reference) and can be used for the expression of ADA in the form of soluble protein in the cytoplasm Pichia pastoris.
Suitable promoter sequences in yeast vectors include the promoters for 3-phosphoglycerate (Hitzeman et al., 1980, J. Biol. Chem., 255:2073) or other glycolytic enzymes (Hess et al., 1968, J. Adv. Enzyme Reg., 7:149; Holland et al., 1978, Biochemistry, 17:4900), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, provideservices, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, providenza, triazolopyrimidine, phosphoglucomutase and glucokinase. When constructing suitable expression plasmids termination sequences associated with these genes are also inserted into the expression vector, ligera their 3'-end expressed sequence to provide polyadenylation of the mRNA and termination of transcription. Other promoters, which have the additional advantage of controlled growth conditions transcription include the promotor region of the alcohol dehydrogenase 2, sociotherapy C, acid phosphatase, enzymes of degradation related to the metabolism of nitrogen, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the utilization of maltose and galactose. Suitable is any plasmid vector containing compatible with yeast promoter, the site of replication initiation and termination sequences.
The site of initiation of replication may be provided either by construction of a vector comprising an exogenous site initiation, for example, which can be derived from SV40 or other viral source (for example, virus polyoma, adenovirus, VSV, BPV), or may be provided by the mechanism of chromosomal replication in the host cell. If the vector is integrated into the chromosome of the host cell, the latter is often sufficient. Other useful items plasmids can include expressing genes encoding protein chaperones, preliminaires or protein-dyslipidemias.
C. Polymer conjugates
In another aspect of the invention mutein HELL, such as proteins Ser74-rbada (SEQ ID NO: 1) and Ser74-rcad (SEQ ID NO: 3), anywhereman with a suitable polymer to obtain a polymer conjugates.
In preferred aspects mutein HELL polypeptide anywhereman with essentially panthenyl polymer, preferably polyalkyleneglycol (PJSC).
HELL-polymer conjugate is generally correspond to the formula (I):
in the cat the swarm
(ADA) is a recombinant mutein adelaideans or its active fragment;
NH - is an amino group of the amino acids present in mateine HELL that serves to attach to the polymer;
(z) is a positive integer, preferably from about 1 to about 80, more preferably from about 5 to about 80, most preferably from about 11 to about 18; and
R includes essentially neoantigenic polymer residue that is attached to the HELL reversible or irreversible way.
In more preferred aspects, the polymers include polyethylene glycol (PEG), PEG may be linear, branched or multipath. The polyethylene glycol has the General formula:
where (n) is a positive integer, preferably from about 10 to about 2300, more preferably from about 40 to about 2300. The average molecular weight of the polymers is in the range from approximately 2,000 to approximately 300,000 Yes. More preferably, the polymers have an average molecular weight of from about 4000 Da to approximately 45 000 Da, even more preferably from 4,000 Da to about 20000 Da. Most preferably, the PEG has molecularmass approximately 5000 daltons. Also discusses other molecular weight in order to meet the needs of the specialist.
In the alternative part of the residue of polyethylene glycol (PEG) of the invention can be represented by the structure:
Y11and Y13independently represent O, S, SO, SO2, NR13or communication;
Y12represent O, S or NR14
R11-14independently selected from hydrogen, C1-6-alkyl, C2-6-alkenyl, C2-6-quinil, branched C3-19-alkyl, C3-8-cycloalkyl, substituted C1-6of alkyl, substituted C2-6-alkenyl, substituted C2-6-quinil, substituted C3-8-cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C1-6-heteroalkyl, substituted C1-6-heteroalkyl, C1-6-alkoxy, aryloxy, C1-6-heteroatomic, heteroaromatic, C2-6alkanoyl, arylcarbamoyl, C2-6-alkoxycarbonyl, aryloxyalkyl, C2-6-alkanoyloxy, arylcarboxylic, substituted C2-6alkanoyl, substituted arylcarbamoyl, substituted C2-6-alkanoyloxy, substituted aryloxyalkyl, substituted C2-6-alkanoyloxy and replaced arylcarboxylic;
(a11), (a12) and (b11) are independently zero or a positive integer, preferably 0-6, more preferably 0, 1 or 2; and
(n) is an integer from about 10 to about 2300.
For example, PEG can be functionalized as a non-limiting manner:
R11, R15and R16independently researched the Simo selected from H, C1-6-Akilov, arrow, substituted arrow, aralkyl, heteroalkyl, substituted heteroalkyl and substituted C1-6-Akilov;
(m) is zero or a positive integer, and preferably is 1 or 2;
Y14is O or S; and
(n) represents the degree of polymerization.
In these aspects, the polymer (R-group) includes a blocking group, i.e. the group that is located on the end of the polymer. The blocking group can be selected from any of the NH2HE, SH, CO2H, C1-6-Akilov, preferably methyl, with the listed group known medium-sized specialists in this field.
In another aspect, the polymer portion of the conjugate may be the part that contains the set of points for attachment of HELL. In the alternative to HELL can be attached many molecules of PEG.
Pharmacokinetic and other properties Paglierani HELL can be adjusted if necessary depending on the desired clinical application by changing the molecular weight of PEG, chemical linkers, and the ratio of PEG chains to the enzyme.
In these aspects of HELL can be attached to panthenol polymer a reversible or irreversible way through various linkers known in the art.
Reversible-linked polymer system m which may be based on the cleavage of the benzyl or lactonization trimethylene castle. Activated polymer linkers reversible-linked polymer systems can be obtained in accordance with U.S. patents (the present applicant) 6180095, 6720306, 5965119, 6624142 and 6303569, the contents of which are incorporated into this description by reference. In alternative HELL-polymer conjugates obtained using certain bitin polymer residues, such as described in U.S. patent 7122189 and 7087229, as well as in applications for U.S. patent 10/557,522, 11/502,108 and 11/011,818 (all present applicant), hereby incorporated into this description by reference. Others considered reversible-linked polymer systems are also described in PCT/US07/78600, the content of which is incorporated into this description by reference.
Illustrative examples of reversible-related or irreversibly-bound HELL-polymer conjugates, described in the present application is described in application for U.S. patent 60/913,039, the contents of which are incorporated into this description by reference.
Conjugation with the polymer preferably is a reaction Paglierani, while such reactions are well-known medium-sized specialists in this field. Briefly, mutein rbada or rcade, reacts with the activated polymer, forming a HELL-polymer conjugate. In this regard, can be used a large variety of activated or functionalized polyethylene glycols, including op the sledge, for example, in U.S. patents (the present applicant) 5122614, 5324844, 5612460 and 5808096 (succinimidylester-activated polyethylene glycol (SC-PEG) and similar activated PEG), U.S. patent 5349001 (PEG-activated cyclic emitional), U.S. patent 5650234, as well as other known medium-sized specialists in this field. A description of each of the foregoing documents is incorporated into the present application by reference. Cm. also activated polymers supplied by the company Nektar/Shearwater Polymers. Average professionals in this field can use various activated forms of the polymers to attach without much experimental work.
Average experts in this field will appreciate that such conjugation reaction is usually carried out in a suitable buffer using a molar excess (several times) of activated PEG. Some preferred conjugates obtained with linear PEG molecules, such as the above SC-PEG, can contain, on average, from approximately 10 to approximately 80 PEG chains on the enzyme molecule HELL. Thus, in this case, can be used in molar excess of several hundred times, for example, 200-1000x. The molar excess is used to branched PEG and PEG attached to the enzyme, usually below and can be determined using techniques described in the patents and patent applications, describing the same that are mentioned below in this application.
In these aspects polyalkylene anywhereman with the protein using a chemical linker, including, for example, succinimidylester, thiazolidindiones, urethane and amide linkers. Polyalkylene preferably covalently attached to the Epsilon-amino group of Lys in HELL, although the prior art known to other sites of covalent attachment. HELL-polymer conjugate can include at least 5 chains of polyethylene glycol is attached to the Epsilon-amino groups Lys in the enzyme and in the alternative may include approximately 11-18 PEG chains attached to the Epsilon-amino groups Lys in the enzyme.
Although HELL conjugated with from about 11 to about 18 PEG molecules on the enzyme molecule, through lysine connection, the ratio of PEG-ADA and may be different for the purpose of modifying the physical and kinetic properties of the combined conjugate to fit any specific clinical situation.
From the above it is obvious that additional aspects of the invention include any commercially available or known activated PEG, or similar polymer for conjugation with an enzyme HELL, or its fragment, in order to ensure the conjugates used in the methods of treatment described in this the overall application. See, for example, the directory Nektar Advanced Pegylation 2004 (Nektar, San Carlos, California), fully incorporated into the present description by reference.
Activated PEG can include linear, branched or U-PEG derivatives, such as described in U.S. patents 5681567, 5756593, 5643575, 5919455, 6113906, 6566506, 6153655, 6395266 and 6638499, 6251382 and 6824766 (also included here in the present description by reference). Unlimited list of such polymers corresponds to polymer systems (i)-(vii) with the following structures:
Y61-62independently represent O, S or NR61;
Y63represents O, NR62, S, SO or SO2;
(w62), (w63) and (w64) independently represents 0 or a positive integer, preferably from about 0 to about 10, more preferably from about 1 to about 6;
(w61is 0 or 1;
MPEG is a methoxy-PEG,
where PEG is defined above, and the total molecular weight of the polymer is from about 2,000 to about 100000 daltons; and
R61and R62independently represent the same groups that can be used as R11.
In addition, it should be understood that in addition to the polymers PEG-based can also is used many other polyalkyleneglycol. For example, the conjugates of the present invention can be obtained by methods which include the transformation of multibeam PEG-HE and star-shaped PEG" products, such as described in the listing Corporation Shearwater "Polyethylene Glycol and Derivatives for Biomedical Application", 2001. Cm. directory NOF Corp. Drug Delivery System catalog. Ver. 8, April 2006. The content of each of these directories included in the present description by reference. Multibeam polymers contain four or more polymer chains, and preferably four or eight polymer chains. In order Pogranichnaya illustration, the remainder of the multibeam polyethylene glycol (PEG) can have the formula:
(x) is 0 and a positive integer, that is, from about 0 to about 28; and
(n) is the degree of polymerization.
In one specific embodiment, the present invention multibeam PEG has the following structure:
where (n) is a positive integer. In one preferred variant of the invention, the polymers have the full molecular weight of from about 2000 Da to about 100000 Da, and preferably from 4,000 Da to 45,000 Da.
In another specific embodiment, the multibeam PEG has the following structure:
in which n is a positive integer. In one preferred variant of the invention, the polymers have the full molecular weight of from about 2000 Da to about 100000 Da, and preferably from 4,000 Da to 45,000 Da.
The polymers can be converted into activated appropriately polymer using activation techniques described in U.S. patent 5122614 or 5808096. In particular, the PEG can have the formula:
(u') is an integer from about 10 to about 570, so it is preferable to provide polymers having the full molecular weight of from about 2000 Da to about 100000 Da, and preferably from about 4000 Da to about 45000 Yes; and to 3 the end parts of the balance keeravani the stands or other lower alkyl.
In some preferred embodiments, the implementation of all 4 chains PEG transformed into a suitable functional groups, i.e., IC and so on, in order to facilitate the joining of the recombinant protein. Such compounds to conversion include:
In the most preferred aspects of the invention the activated polyethylene glycol is polietileno is l, which provides the formation of urethane links or amide bond with the protein.
In other alternative aspects of the activated polymers can be used sterically hindered linker on the basis of ester. Cm. PCT/US07/78593, entitled "Polyalkylene Oxides Having Hindered Ester-Based Biodegradable Linkers", the content of which is incorporated into the present application by reference. For example, non-limiting list of such compounds includes:
where (u) is an integer, preferably providing polymers having full molecular weight of from about 2000 Da to about 100000 Yes.
In one preferred embodiment, the PEG conjugate includes:
where (u) is an integer providing a polymer portion having a molecular weight of from about 2000 Da to about 100000 Da, and preferably from about 4000 Da to about 45000 Yes, more preferably about 5000 Da.
Suitable polymers significantly differ in weight. The polymers having srednecenovogo molecular weight ranging from about 2000 to about 100000, usually included in the scope of the present invention. Molecular weight of from about 4000 to about 45000 are prefer the elegance, and from 5,000 to approximately 12000 is the most preferred. Included polymeric substances are also preferably are soluble in water at room temperature. A non-limiting list of such polymers include homopolymers of polyalkyleneglycol, such as polyethylene glycol (PEG) or polypropylenglycol, polyoxyethylene polyols, copolymers and block copolymers, provided that there is a water-solubility of the block copolymers. In addition to MPEG can also be used polymers with terminal C1-4-alkilani.
Methods for producing polymers having terminal carboxyl groups, with high purity, as described in application for U.S. patent 11/328,662, the contents of which are incorporated into the present application by reference. Methods will include first obtaining a complex tertiary alkyl ester of polyalkylated with subsequent conversion to the corresponding carboxylic acid derivative. The first stage of obtaining PAO carboxylic acids of the method involves the formation of intermediate compounds, such as t-butyl ether polyalkylene-carboxylic acid. This intermediate compound is formed in the reaction PJSC with t-butylchloroformate in the presence of a base, such as t-butyl potassium. After the formation of the intermediate t-butyl ester derived polyalkyloxy-carboxylic acid can the be easily obtained with purity, greater than 92%, preferably greater than 97%, more preferably greater than 99% and most preferably with a purity in excess of 99.5%.
In other alternative aspects of the polymers having terminal amino groups, can be used to get the HELL conjugates. Methods for producing polymers containing terminal amines with high purity, described in patent applications U.S. 11/508,507 and 11/537,172, the contents of which are incorporated by reference. For example, polymers having azides, interact with the reducing agent on the basis of a phosphine, such as triphenylphosphine, or with alkali metal borohydride, such as NaBH4. In alternative polymers, including leaving groups react with the salts protected amines, such as potassium salt of methyl-tert-butylmethacrylate (KNMeBoc) or potassium salt of di-tert - butylmethacrylate (KNBoc2and then unprotect a protected amino group. The purity of polymers containing terminal amines obtained by the following methods, in excess of about 95% and preferably greater than 99%.
Branching, which provide polymers of the patent 6,153,655 above, allows secondary or tertiary branching as a way of increasing the load of the polymer and a biologically active molecule from a single attachment point. It should be understood that p is storymy in water, the polymer can be functionalized with the purpose of attaching a bifunctional cross-linking groups, if necessary, without undue experimental work.
Polymeric substances in the present invention, preferably soluble in water at room temperature. A non-limiting list of such polymers include homopolymers of polyalkyleneglycol, such as polyethylene glycol (PEG) or polypropylenglycol, polyoxyethylene polyols, copolymers and block copolymers, provided that there is a water-solubility of the block copolymers.
Alternatively, the polymers based on the PSC can be used practically non-allergenic materials such as dextran, polyvinylpyrrolidone, polyacrylamides, such as HPMA (hydroxypropylmethacrylamide), polyvinyl alcohols, polymers based on carbohydrates, copolymers of the above polymers, and so on Average the experts in the art are aware that the above list is only illustrative, and that covers all polymeric materials, which are described in this application. In the framework of the present invention, the term "essentially or virtually non-allergenic" means all materials which, as is known from the prior art, non-toxic and do not cause appreciable immune response in mammals.
The specialist will be able to appreciate that mutein HELL of the present invention will readily find application in a clinical setting for treatment is any disease or impairment, susceptible enzyme HELL. Such disease or disorder is a disease or disorder that responds to the reduced levels of adenosine or deoxyadenosine in tissues or blood. Such disease or disorder may include, for example, DID, lung diseases, such as asthma and cancer, which react at reducing local or systemic levels of adenosine or deoxyadenosine. More detailed information regarding the application of HELL in the treatment of tumors or cancers are listed in the filed on the same date with the present application the application (the present applicant) U.S. patent ________ (which claims the priority of provisional patent application U.S. 60/913,039), entitled "Enzymatic anticancer therapy" and is fully incorporated by reference into the present application. therapeutic agent can be, for example, mutein rcade or mutein rbada. Preferably, therapeutic mutein happy is conjugated to the polymer, as described above, for example, Paglinawan. Dose of HELL or HELL-polymer conjugate is individualized depending on the clinical response of the tumor and profile of side effects at the individual - animal or human. In the examples below, the highest dose is the maximum dose that can be transferred the tee.
For example, Adagen®supplied commercially in the form A bullish HELL/ml, This corresponds to 2000 U/kg for mice weighing approximately 25 g after administration of 0.2 ml Adagen®. Of course, the specialist will be able to appreciate that the dose of HELL-polymer conjugate can also be adjusted depending on the size of the specific polymer, the chemical linker and valence. For example, the dosage for a polymer conjugate comprising two or more enzymes of HELL on the polymer, is regulated according to the units of HELL in ml solution of any particular polymer conjugate of HELL.
While ensuring HELL or conjugate HELL with PEG via injection, the optimal dose range is preferably set in the control plasma. In General, it is desirable to provide the recipient of such a dosage that supports the ADA activity in plasma (minimum levels) in the range from approximately 10 to 100 çmol/h/ml, preferably from about 15 to about 35 çmol/h/ml (in the analysis at 37°C), and demonstrates the decrease of erythrocyte adenosine, i.e dATP to ≤ approximately 0,001-0,057 μmol/ml, preferably approximately 0.005 to about 0.015 μmol/ml Packed erythrocytes, or ≤ approximately 1% of the total erythrocyte adenosine (i.e., the ATP content + DTF), relative to normal levels of adenosine, pristerene in the sample, taken before the injection. Normal value datf is less than approximately 0.001 µmol/ml
Dose based on the amount of enzyme varies, for example, from about 0.1 U/kg to about 30 U/kg, or higher, preferably from about 0.5 U/kg to about 20 U/kg, and more preferably from about 0.5 U/kg to about 12 U/kg (i.e., per kg weight of the patient), as for example, from about 0.5 U/kg to about 5 u/kg of Total weekly dose can be up to 40 U/kg or more, if tolerated by the recipient. Allowed subsequent dose increases in the amount of 5 u/kg/week, up to a maximum single dose of 30 U/kg or more, if this dose is tolerated by the recipient. Usually after weekly injections ADAGEN®at a dose of 15 U/kg average minimum level of ADA activity in plasma ranges from 20 to 25 çmol/h/ml
It should be noted that the dose of 100 U/kg mouse is the dose equivalent clinical pediatric dose approximately 12 U/kg
Detailed information regarding the dosage of HELL known in the prior art and are provided in the insert with instructions for medical use of the drug ADAGEN®(Enzon, Inc.), the content of which is incorporated into the present application.
The following examples serve to provide a more complete understanding of izopet the tion, but in no way limit the actual scope of the invention.
The design of gene-expression strain of E. Coli expressing recombinant HELL man with the replacement of the Cys/Ser at position 74 of the Mature protein
The published amino acid sequence with a length of 363 A.K. adelaideans human (GenBank NP_000013 included in the present application by reference) were analyzed for the presence of cysteine codons. Five provisions in Mature (derived N-terminal Met) the polypeptide encode cysteine (C74, C 12, C153, C168, C261). In constructed and modified gene expressing the HELL man, only one of these five codons cysteine (Cysteine 74, TGC) was replaced by a serine codon (TCC) (75 position in the translated protein). Certain polypeptide sequence (see SEQ ID NO: 3) provided the Corporation Blue Heron (Bothell, Washington, USA) for the synthesis of a new full-size gene comprising codons optimized for expression in E. coli, using standard chemical synthesis of overlapping oligonucleotide fragments. Briefly, the sequence is optimized for bacterial expression in accordance with the standard bacterial codons used for Escherichia coli K12 using data for codons described Grantham, R. et al.; 1981, "Codon catalogue usage in genome strategy modulated for gene expressivity," Nucleic Acid es. 9:r43-r47, and Lathe, R. 1985, "Synthetic oligonucleotide probes deduced from amino acid sequence data, Theoretical and practical considerations," J. Mol. Biol.; 183:1-12.
Then the corresponding sequence of the RNA was analyzed on the subject of the formation of hairpin structures or loops and subjected to computing the minimum free energy. Flanking the restriction sites NdeI and BamHI was inserted in the ends of the gene. After cleavage of synthetic DNA by restriction enzymes NdeI and BamHI 1,1 TPN gene ligated using T4 DNA ligase in the plasmid vector pET-28a (Novagen), which was also split these two enzymes. The recombinant plasmid was introduced into E. coli strain BLR (DE3) or HMS 174 (DE3) by electroporation using a BTX Electro Cell Manipulator 600, according to the manufacturer's instructions. Transformed culture were sown on plates with LB agar containing kanamycin (15 μg/ml) for selection of colonies containing the plasmid pET-28a/ADAcysSer (marked ADAc75s/pET28a:BLR (DE3) or ADAc75s/pET28a:HMS174 (DE3)). Nucleotide sequence variants of the gene HELL was confirmed through analysis of DNA sequences using the ABI Prism 310 Genetic Analyzer using Big Dye Terminators. The DNA sequence encoding the open reading frame Ser74-rcad corresponded to SEQ ID NO: 4.
Then, the selected colonies were purified, Parasiva on the Cup, and analyzed for expression induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG), in which the LB ed by standard methods, such as described in the ninth edition Novagen pET System Manual, incorporated by reference in this application.
Investigated several parameters of induction, including time, temperature and concentration of inducer. The preferred conditions of induction, including the induction of 50 μm IPTG for 12 hours at 25°C, provided the products for HELL and high levels in the cytoplasm of bacterial hosts in the amount of approximately 20% of the total protein of the cells. Downregulation of protein HELL, have been analyzed by electrophoresis in LTO-page, had the desired molecular weight of approximately 40 kDa (data not shown).
The design of gene-expression strain of E. Coli expressing recombinant bovine HELL with the replacement of the Cys/Ser at position 74 of the Mature protein
Purified Mature protein HELL, obtained from preparations of the intestinal tract of cattle, is a protein with a length of 356 amino acids, without N-terminal methionine, and without the six C-terminal residues predicted from the cDNA sequence (GenBank NP_776312, incorporated by reference in the present application). Amino acid sequence of bovine ADA analyzed for the presence of cysteine codons. Five positions in the Mature polypeptide coding for a cysteine (C74, C152, C153, C168, C261). In constructed and modified synthetic gene bullish HELL only one pointed to by the x five positions of cysteine (cysteine 74) was replaced by a serine residue. This is performed by introducing a serine codon (TCC) instead of normal cysteine codon at position 74 of the Mature polypeptide (or position 75 of product broadcast). Gene optimized by codons for expression in E. coli.
Briefly, a specific polypeptide sequence (see SEQ ID NO: 1) has provided the company BioCatalytics for the synthesis of a new full-size gene comprising codons optimized for expression in E. coli, using its own methods BioCatalytics, which include chemical synthesis of overlapping oligonucleotide fragments. How BioCatalytics described in more detail in U.S. patent 6366860, the contents of which are fully incorporated by reference into the present application.
Expression of bovine ADA analyzed in several expression systems. For example, in the ends of the gene were introduced flanking the restriction sites NdeI and BamHI. After cleavage of synthetic DNA by restriction enzymes NdeI and BamHI gene 1,1 TPN ligated using T4 DNA ligase in the plasmid vector pET-9d (Novagen), which was also split these two enzymes. The recombinant plasmid was introduced into E. coli strain BLR (DE3) or HMS 174 (DE3) by electroporation using a BTX Electro Cell Manipulator 600, according to the manufacturer's instructions. Transformed culture were sown on plates with LB agar containing kanamycin (15 μg/ml) for selection of colonies containing their plasmid pET-9d/bADA (marked bADA/pET9d:BLR(DE3) or bADA/pET9d:HMS 174(DE3)). Nucleotide sequence variants of the gene HELL was confirmed through analysis of DNA sequences using the ABI Prism 310 Genetic Analyzer using Big Dye Terminators. The DNA sequence containing the open reading frame Ser74-rcad shown in SEQ ID NO: 2.
Then, the selected colonies were purified, Parasiva on the Cup, and analyzed on the subject of IPTG-induced gene expression in LB medium by standard methods such as described in the ninth edition Novagen pET System Manual. Analyzed several parameters of induction, including time, temperature and concentration of inducer. The preferred conditions of induction, including the induction of 0.3% lactose for 12 hours at 37°C, provided the products for HELL and high levels in the cytoplasm of bacterial hosts in the amount of approximately 20% of the total protein of the cells. The product of HELL, have been analyzed by electrophoresis in LTO-page, had the desired molecular weight of approximately 40 kDa.
Cleaning mutein rcade
Cleaning mutein rcade were performed using 3-stage chromatographic purification, developed by Enzon. Cultivation of bacterial cells was performed with E. coli strain expressing the protein Rada with synthetic gene into plasmid pET28a (Novagen) in the cell-master of HMS 174(DE3). In minimal medium with glycerol included rifampicin (200 μg/ml) and kanamycin μg/ml) with addition of yeast extract (30 g/l), and cells were grown at 28°C until OD60011, after which was added inducer IPTG to a final concentration of 5 mm. After 40 hours (OD600~110) the cells were collected by centrifugation and frozen at -20°C. Briefly, thawed cell paste (50 g) resuspendable in 1800 ml of a buffer containing 10 mm Tris [trihydroxystilbene], 1 mm DTT, pH 8.0, and homogenized at 1200 rpm for 10 seconds on Tempest Virtis (SentryTM,A Microprocessor, Boston, MA). The resulting suspension was passed through a stainless steel sieve with openings of 250 μm, No. 60, W.S Tyler) to remove large particles. Homogeneous cell suspension is homogenized using a homogenizer high pressure in 3 cycles when 103,4 MPa (15000 psi) (set-cooled ice) (MicroFluidizer, Microfluidics Corp., Model#110Y, Boston, MA). At the end of microgametogenesis 200 ml of the same above the buffer used for washing installation, and the resulting solution was combined with the above suspension. Soluble protein from cell lysates were isolated by centrifugation at 16000 rpm for 40 minutes at 4°C (Sorvall®RC 5C plus, the rotor SLA-1000). The supernatant was collected carefully to avoid unwanted mixing. The pH was brought to 8.0, and then added 1 mm MgCl2and 20 mg/ml Gnkazy, and incubated at room temperature for 2 hours. Then the pH was brought to 6.5 1 N. HCl. The second centrifugation, p is bodily as well as described above, the supernatant was collected and added EDTA to a concentration of 2 mm, followed by filtration using a 90 mm filter device Nalgene®. The volume of the filtered supernatant was 500 ml, the concentration of total protein by BCA method (with bicinchoninic acid) was 8.5 mg/ml
Cell extract (100 ml) was brought to pH of 7.2 and 4.5 MS/cm was applied to a HiTrap®DEAE ff (ff meaning high flow rate) in 20 mm Bis-Tris, 20 mm NaCl, pH 6.5, and suirable in 20 mm Bis-Tris, 500 mm NaCl, pH 6.5. Peak fractions were analyzed using enzyme analysis and electrophoresis LTO-page, after which was added ammonium sulfate to a concentration of 1.5 M in 20 mm NaHPO4, pH 6.5 and applied to a column HiTrap Phenyl ff. Protein was suirable in gradient buffer for applying and 20 mm NaHPO4, a pH of 6.5. The peak fraction (55 ml, 0.4 mg/ml) was subjected to diafiltration against buffer containing 20 mm NaHPO4, 1 mm EDTA, 1 mm DTT, pH 6.5 and was applied to a HiTrap SP-Sepharose ff and suirable 20 mm NaHPO4, 500 mm NaCl, 1 mm EDTA, 1 mm DTT, pH 6.5. The collected fraction contained the purified protein HELL (77 ml, 0.1 mg/ml).
Purification of recombinant bovine protein HELL
Cleaning mutein rbada expressed a clone of Example 2 were performed using 3-stage chromatographic purification, developed by Enzon. Briefly, 200 g of thawed cell paste (obtained from Blue space hereon is whether Biocatalytics, respectively), which was stored at -80°C, resuspendable in 1800 ml of buffer containing 20 mm Bis-Tris, 1 mm EDTA, pH 7.4 and homogenized at 1200 rpm for 5 minutes at Tempest Virtis (SentryTM, A Microprocessor, Boston, MA). The resulting suspension was passed through a stainless steel sieve with openings of 250 μm, No. 60, W.S Tyler) to remove large particles. Homogeneous cell suspension homogenized using a homogenizer high pressure in 3 cycles when 103,4 MPa (15000 psi) (set-cooled ice) (MicroFluidizer, Microfluidics Corp., Model#110Y, Boston, MA). At the end of microgametogenesis 200 ml of the same above the buffer used for washing installation, and the resulting solution was combined with the above suspension. Soluble protein from cell lysates were isolated by centrifugation at 7100 rpm (12000×g) for 60 minutes at 4°C (Avanti J-201, Beckman Coulter rotor JLA8.1000). The supernatant was collected carefully to avoid unwanted mixing.
Removal of nucleotides in a given cell extract to the above supernatant was added polyethylenimine (PEI) (to a final concentration of 0.15%, mass/V) and thoroughly mixed for 10 minutes and the resulting cell extract was left overnight at 4°C. the Precipitate from the specified night of the sample was removed with centrifugation at 7100 rpm (12000×g) for 60 minutes at 4°C (Avanti J-201, Beckman Coulter rotor JLA8.1000). Exactly the same supernatant was carefully collected, to avoid unwanted mixing. In order to facilitate binding of HELL in the first column of this cell extract was slowly added 10%PEG4600, and the pH of the resulting cell extract was slowly brought up to 6.5 1 N. NaOH 1 N. HCl. Then the supernatant was again centrifuged at 7100 rpm (12000×g) for 60 minutes at 4°C (Avanti J-201, Beckman Coulter rotor JLA8.1000) before applying to the next column.
Cell extract was applied onto a pre-equilibrated column Capto Q (cat. 17-5316-01, GE Healthcare, Piscataway, NJ). Layer volume 350 ml column XK-50 pre-saturated buffer containing 20 mm Bis-Tris, 1 mm EDTA, pH 6.5. Before elution of HELL from the column equilibrating buffer containing 80 mm NaCl, to remove impurities first performed elution with 60 mm and 70 mm NaCl. The elution profile was analyzed for ADA activity, using electrophoresis in LTO-SDS page, Western blotting and RP-HPLC.
After column Capto Q, one after the other held two purification using hydrophobic interaction chromatography (HIC), to further improve the purity of the protein. The first HIC-purification was performed on octyl-sepharose 4FF (cat. 17-0946-02, GE Healthcare, Piscataway, NJ). Combined fractions of HELL after column Capto Q directly besieged powder of ammonium sulfate at a concentration of 1.5 M (NH4)2SO4, and brought the pH to 6.5. The filtered sample (Nalgene Nunc, number ka is. 540887, MEMB 0.2 PES, Rochester, NY) was applied on the first HIC column, pre-equilibrated with 1.5 M (NH4)2SO4, 20 mm potassium phosphate, 1 mm EDTA, pH 6.5 (layer volume 150 ml, XK-50, GE Healthcare, Piscataway, NJ). Protein HELL was suirable in a gradient of ammonium sulfate, and the profile of the purity of the obtained eluate was determined by electrophoresis in LTO-page and RP-HPLC. Protein HELL in the fractions of the first column HIC were United and brought the concentration of (NH4)2SO4up to 1 M, and then directly applied to the second column HIC (layer volume 150 ml, XK-50, HIC Phenyl HP, number cat. 17-1082-01, Piscataway, NJ)pre-equilibrated 1 M (NH4)2SO4, 20 mm KH2PO4-K2HPO4, 1 mm EDTA, pH 6.5. HELL was suirable in a gradient of ammonium sulfate from 1 M to 300 mm 20 mm KH2PO4-K2HPO4, 1 mm EDTA, pH 6.5. The purity of HELL in the obtained fractions were analyzed by electrophoresis in LTO-page and RP-HPLC. Then peeled rbada or rcade was absoluely and concentrated using LabScale™ TFF (BioMax Membrane 5, Bedford, MA) against the buffer for storage (for example, 100 mm sodium phosphate, 1 mm EDTA, pH 6.5).
Analyses of stability rbada and Ser74-rbada
The following tests were performed to demonstrate that the stability rbada when oxidative degradation was really enhanced by mutations replacing cys74/ser74. For analysis the and stability of the used samples of recombinant bovine ADA (rbad) and recombinant bovine HELL with mutation cys74/ser74 (Ser-74-rbada) in concentrations of approximately 0.5 mg/ml in sodium phosphate buffer (pH 7.8). Stability was monitored using reverse-phase HPLC (RP-HPLC)using UV detection at 220 nm and mass spectrometric detection (mass spectrometer Micromass Q-TOF with ionization elektrorazpredelenie). The conditions of HPLC were as follows:
|Column:||Bond 300 SB-C8 (Agilent, 250×4.6 mm, pore size of 300 angstroms, particle size 5 microns).|
|The mobile phase A:||0,1% triperoxonane acid in water.|
|The mobile phase B:||0,1% triperoxonane acid in a mixture of acetonitrile/water (80/20, V/V).|
|Gradient:||Time||% mobile phase B|
|The temperature of the column:||40°C|
|Flow rate:||1.0 ml/min|
|Enter amount:||50 µl|
The purity of the compounds was determined using RP-HPLC analysis in the original beginning of the analysis of stability and at different points in time, including 4, 8 and 17 days after the start of the analysis. It should be noted that the samples rbad (not mutein) prior to this study were stored for approximately two months and has already undergone some decomposition. Sample Ser74-rbada was prepared recently and was relatively clean. However, in this study, the analyzed parameter was the difference in purity between the initial time and after 17 days of incubation at 25°C.
As shown in Table 1, the purity rbada amounted to 83.7% at the initial moment of time and decreased to 66,1% after 17 days, which indicates that 17,6% rbada corrupted during a specified period of time. Mass spectrometric analysis of chromatographically separated peaks showed that the main product of degradation, eluruumis on 31,851 minute and held 30,5% of the area of the chromatogram, had a lot on 32 Da higher than rbada. This change in mass corresponds to the joining rbada 2 atoms of oxygen with the formation of the product degrades and, containing sulinowo acid instead of the free cysteine at position 74 rbada. Weight of product degradation, which had a minor peak, aliremove on 32,538 minute, consistent with the accession 1 atom of oxygen to rbada with the formation of product degradation, containing Sultanova acid instead of the free cysteine at position 74 rbada. Ser74-rbada that contains a serine residue that replaces reactive cysteine residue 74, showed a slight degradation in 17 days, with the purity of 17 days (97,9%) actually has not changed in comparison with the purity on the initial moment of time (97,2%). This proves that the cysteine 74 indeed is a source of oxidative degradation, which occurs in rbada, and mutation with the replacement of the specified residue to serine, which is not prone to oxidation, prevent such decomposition.
|Stability rbada and Ser74-rbada in sodium phosphate buffer|
(pH 7.8) at 25°C
|Time||% purity when using RP-HPLC|
|Initialtd align="center"> 83,7||97,2|
The use of Malinov HELL in the treatment of ADA-deficient patients with TCID
Described mutated enzymes HELL used in therapeutic schemes that currently apply to ADAGEN®. Ser74-rb or rcade can be modified by conjugation with polyethylene glycol (PEG), for example, 11-17 PEG 5 kDa polymers on protein HELL. Paglierani drugs mutein HELL is injected in a sterile solution of sodium chloride at a pH of 7.2-7.4 and at a concentration of approximately 250 units per milliliter. Targeted mutein HELL is administered to patients parenterale, for example, intramuscularly. Patients with indications for such therapy include patients with severe combined immunodeficiency caused by insufficient activity of HELL. The introduction of PEG-ADA mutein usually performed every seven days according to the scheme dosing 10 U/kg for the first dose and 20 U/kg per week for subsequent doses. Ser74-RB / RF PEG-ADA stored at 2-8°C in water dissolve the e with a single dose vial with a volume of 1.5 ml. The dosing scheme is designed to ensure the levels of ADA activity in plasma is of the order of 15-35 çmol/h/ml (in the analysis at 37°C) and lower erythrocyte dATP to ≤0.005 to 0.015 μmol/ml Packed erythrocytes.
Getting Paglierani Ser74-rbada through education
SC-PEG (N-hydroxysuccinimide-activated polyethylene glycol, 0,084 mmol) was added to a solution of Ser74-rbada (0,00027 mmol) in 3 ml of sodium phosphate buffer (0.1 M, pH 7.8) with mild stirring. The solution was stirred at 30°C for 30 minutes. To control PEG-conjugation used GPC-column (Bond GF-450). At the end of the reaction (as evidenced by the lack of native enzyme) and the mixture was diluted with 12 ml of buffer for the preparation of compositions (0.05 M sodium phosphate, 0.85 per cent sodium chloride, pH 7.3), and then subjected diafiltration using Centriprep concentrator (Amicon) to remove unreacted PEG. Diafiltration kept at 4°C for as long as needed, until the free PEG was no longer detected by mixing equal quantities of filtrate and 0.1% PMA (polymethacrylic acid in 0.1 M HCl).
Getting Paglierani Ser74-rcad through education
The reaction of SC-PEG (0,084 mmol) and Ser74-rcad (0,00027 mmol) was performed using a t the x same conditions as described in Example 7.
Getting Paglierani Ser74-rbada through education
SS-PEG (N-hydroxysuccinimidyl-activated polyethylene glycol, 0,084 mmol) was added to a solution of Ser74-rbada (0,00027 mmol) in 3 ml of sodium phosphate buffer (0.1 M, pH 7.8) with mild stirring. The solution was stirred at 30°C for 30 minutes. To control PEG-conjugation used GPC-column (Bond GF-450). At the end of the reaction (as evidenced by the lack of native enzyme) and the mixture was diluted with 12 ml of buffer for the preparation of compositions (0.05 M sodium phosphate, 0.85 per cent sodium chloride, pH 7.3), and then subjected diafiltration using Centriprep concentrator (Amicon) to remove unreacted PEG. Diafiltration kept at 4°C for as long as needed, until the free PEG was no longer detected by mixing equal quantities of filtrate and 0.1% PMA (polymethacrylic acid in 0.1 M HCl).
Getting Paglinawan mutein rcade through the formation of amide bond
The reaction of SS-PEG (0,084 mmol) and muteena Rada (0,00027 mmol) was performed using the same conditions as described in Example 9.
1. Selected recombinant adelaideans comprising the polypeptide SEQ ID NO: 1 or a variant polypeptide SEQ ID NO: 1 selected recombinant adelaideans, devarennes polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions, selected from the group consisting of: Gln instead Lys198; Ala instead Thr245; and Arg instead Gly351.
2. Recombinant adelaideans according to claim 1, encoded by the DNA molecule of SEQ ID NO: 2.
3. Conjugate of polyalkylated with adenozindeaminaza to ensure adelaideans-mediated diseases, where adelaideans is recombinant adenozindeaminaza according to claim 1, where the recombinant adelaideans comprises from 11 to 17 chains polyalkylated with a molecular mass of 5 kDa protein of HELL.
4. Conjugate of polyalkylated with adenozindeaminaza according to claim 3, where polyalkylene is polyethylene glycol.
5. Conjugate of polyalkylated with adenozindeaminaza according to claim 4, where the polyethylene glycol anywhereman with recombinant adenozindeaminaza through a linker selected from the group consisting of succinimidylester, thiazolidindiones, urethane and amide linkers.
6. Conjugate of polyalkylated with adenozindeaminaza according to claim 4, where the polyethylene glycol is covalently attached to the Epsilon-amino group of Lys recombinant adelaideans.
7. Conjugate of polyalkylated with adenozindeaminaza according to claim 4, where the polyethylene glycol anywhereman with recombinant adenozindeaminaza through succinimidylester the linker.
8. The method of purification of recombinant adelaideans according to claim 1, including protein purification using ion-exchange x is omatography.
9. The method of purification of recombinant adelaideans according to claim 1, including protein purification using hydrophobic interaction chromatography.
10. Recombinant adelaideans obtained by the method according to item 8.
11. Recombinant adelaideans obtained by the method according to claim 9.
12. The selected DNA, which encodes the recombinant adelaideans having the amino acid sequence comprising SEQ ID NO: 1.
13. The selected DNA, which encodes the recombinant adelaideans according to claim 1.
14. Selected recombinant adelaideans according to claim 1, comprising the polypeptide SEQ ID NO: 1.
SUBSTANCE: bacteria strain Planomicrobium koreense 78k, is extracted from soils and provides for production of site-specific endonuclease PkrI, which recognises and splits both chains of nucleotide sequence of DNA, which contains three or four C5-methylcytosine bases in recognition site 5'-GCNAGC-3' with formation of a single-nucleotide 3'-protruding end. The strain is deposited in the Russian National Collection of Industrial Microorganisms FGUP GosNIIgenetika under the number B-10627. The provided strain may be used for extraction of the new site-specific endonuclease PkrI, which may be applied in detection and splitting of methylated DNA sections.
EFFECT: production of site-specific endonuclease PkrI.
3 dwg, 3 ex
SUBSTANCE: invention represents a bacteria strain Microbacterium testaceum 17B, deposited under the number RNCIM B-10628 in the Russian National Collection of Industrial Microorganisms FGUP GosNIIgenetika, being the producent of the site-specific endonuclease MteI. The invention makes it possible to produce the site-specific endonuclease, which recognises and splits both chains of the nucleotide DNA sequence 5'-G(m5C)G(m5C)∧NG(m5C)G(m5C)-3'/3'-(m5C)G(m5C)GN∧(m5C)G(m5C)G-5', where m5C-5-methylcytosine, with production of the single-nucleotide 5'-protruding end, and does not split the methylated DNA sequence 5'-G(m5C)NG(m5C)-3'/3'-(m5C)GN(m5C)G-5'.
EFFECT: method improvement.
2 dwg, 1 tbl, 3 ex
SUBSTANCE: supernatant containing a recombinant protein with an amino acid sequence of staphylokinase (RPSF) is defrozen. The RPSF is recovered and chromatographically purified on CM-sepharose. The RPSF is chromatographically purified on DEAE-sepharose. Then the RPSF is transferred into 0.3% NaCl solution. It is followed by sterilisation, packaging, lyophilisation and marking of the substance. 20 litres of water for injections are introduced in a stirred reactor 160 l, then 150 g of the substance and 10 l of water for injections are added; the stirrer is actuated (stirring rate is 80 rpm); the stirring procedure is carried out for 15 min; thereafter the stirrer is stopped; the substance is sampled; the pH value of the prepared solution is recorded; pH is 5.5 to 7.5. It is followed by sterilisation filtration of the solution, filling and pre-corking of bottles. Then lyophilisation and final corking are performed. What is offered is the application of the preparation Fortelysin for treating myocardial infraction by the intravenous introduction in dose 15 mg (2235000 IU) with the content of a bottle 5 mg (745000 IU) before the introduction of the preparation is dissolved in 0.9% sodium chloride 5 ml; the preparation is introduced in two boluses 10 mg (1490000 IU) simultaneously and in 30 minutes 5 mg (745000 IU) or at first 10 mg (1490000 IU) in boluses, and the rest 5 mg (745000 IU) in intravenous infusions in 0.9% sodium chloride 50 ml for 30 min.
EFFECT: invention extends the range of drug preparations with fibrinolytic properties.
SUBSTANCE: disclosed is a plasmid which determines synthesis of alkaline phosphatase CmAP, containing a NcoI/SacI-fragment of plasmid pET-40b(+) (Novagen) and a DNA fragment of 1530 base pairs, which contains a chimeric gene consisting of a CmAP gene structural part which is adapted at the N-end for expression in E.coli cells, and a nucleotide which codes a specific sequence for TEV protease. Described is an E.coli Rosetta(DE3) strain which is transformed by said plasmid, and is a producer of chimeric protein, which contains an amino acid sequence of recombinant alkaline phosphatase CmAP. Disclosed is a method of obtaining recombinant alkaline phosphatase CmAP, comprising steps for: incubating said producer strain in a liquid broth LB for 12 hours at 20°C, depositing bacterial cells by centrifuging, disintegration of the cell suspension in a buffer, centrifuging the extract, chromatography of the supernatant fluid on a column with a metal affinity resin, elution of the protein, concentrating active fractions by ultrafiltration, incubation with protease TEV, concentrating the protein solution and extracting the end product by gel filtration.
EFFECT: improved method.
3 cl, 1 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to veterinary, agriculture, medicine, biotechnology and microbiological industry and can be applied for protection from infections, induced by DNA - and RNA- containing viruses. Antiviral combined medication of contact action based on "Betadine", represents mixture of medication "Betadine" with solution of bacterial endonuclease - product of gen nuc A (synonym nuc), in any ratio, and content of povidone-iodine in diluted medication "Betadine" constitutes 0.005 - 0.000005%, and pH of solution of bacterial endonuclease in buffer or salt solution, which contains magnesium cations, constitutes 7-9.
EFFECT: medication has simultaneously reduced irritating effect and high virulicidal activity.
SUBSTANCE: Trichoderma aureoviride Rifai fungus strain is recovered from sea ground collected at depth 25 m in the South China Sea. It is deposited in the Russian National Collection of Microorganisms of G.K.Skryabin Institute of Microbial Biochemistry and Physiology of the Russian Academy of Sciences, registration No. VKM F-4115D. The strain is cultivated with using a nutrient medium not containing sugars. Total yield pf 1,3-β-D-glucanase is 13.5-16 units/mg of protein.
EFFECT: strain is characterised by high yield of 1,3-β-D-glucanase.
1 tbl, 2 ex
SUBSTANCE: claimed is method of determining DNA-hydrolysing activity of molecules and device for its realisation. Method includes adsorption of reaction mixture, which contains DNA and tested substances, on working electrode, modified with carbon nanomaterials. After that, registered and compared are values of DNA oxidation in composition of reaction mixture before standing reaction mixture (control) and after standing reaction mixture (experiment). Increase of DNA oxidation signal testifies to presence of DNA-hydrolysing activity in tested molecule.
EFFECT: invention makes it possible to simplify and reduce terms of determining DNA-hydrolysing activity of molecules, increase selectivity and sensitivity of the test reducing its cost.
8 cl, 6 dwg, 2 ex
SUBSTANCE: strain of bacteria Kocuria rosea 307, isolated from soil and providing a site-specific endonuclease Krol is put forward. Endonuclease Krol recognises and cleaves both strands of nucleotide sequence of DNA 5'-GCCGGC-3'/3'-CGGCCG-5' in which two central cytosines are methylated in position C5 with formation of four-based 5'-protruding ends. For strain cultivation media of the following composition (g/l) is used: peptone - 10, yeast extract - 5, NaCl - 5, distilled water - the rest. Cultivation is carried out at 28°C with aeration up to attaining the stationary stage of growth. Yield of enzyme is 200 u/g of raw biomass, the concentration is 1000 u/ml.
EFFECT: invention yields elevation of enzyme.
2 dwg, 3 ex
SUBSTANCE: invention represents polypeptide, having α-L-arabinofuranosidase activity selected from the following polypeptides: polypeptide with SEQ ID No. 2, polypeptide, amino-acid sequence of which is located between positions 28 and 400 SEQ ID No. 2, fragment of polypeptide with SEQ ID No. 2, having activity of α-L-arabinofuranosidase, polypeptide having activity of α-L-arabinofuranosidase B and expressing 80% identity with polypeptide SEQ ID No. 2. Invention also relates to polynucleotide, which codes this polypeptide, expression cassette and vector, containing polynucleotide, and master organism that contains this polypeptide.
EFFECT: expanded arsenal of mediums for hydrolysis of α-L-arabinofuranosyl links in arabinofuranosyl-oligosaccharide compounds.
9 cl, 6 dwg, 2 tbl, 1 ex
SUBSTANCE: immobilised biocatalyst contains cells of filamentous fungi, which produce pectinases and are included into matrix of gel carrier containing polyvinyl alcohol. Gel carrier represents cryogel of polyvinyl alcohol with macropores of 0.5-5.0 mcm section. Immobilised biocatalyst is produced on the basis of the following components, taken in the following ratio (wt %): cells of filamentous fungi (by dry mass) - 0.001-0.1, polyvinyl alcohol - 8.4÷12.5; aqueous phase - up to 100. Time of biocatalyst application makes 572-744 parts, average productivity of process by pectolytic activity makes 1.05-1.45 Units.ml-1part-1, and maximum pectolytic activity makes 104160-830000 Units.
EFFECT: invention provides for durable preservation of high mechanical strength by immobilised biocatalyst and vitality of filamentous fungi cells having pectolytic activity.
SUBSTANCE: method comprises culturing CHO cell in which the gene of taurine transporter (TauT) was artificially transferred, the DNA encoding the desired polypeptide and the DNA encoding dihydrofolate reductase (DHFR) was introduced in the presence of methotrexate concentration at which 90% or more cells, in which TauT was not introduced die within three weeks after subculturing. From the number of surviving CHO cells the CHO cell is selected which is capable to produce a desired polypeptide with a high yield. The cell prepared in this manner produces a desired polypeptide in higher yield than the CHO cell transfected with DNA encoding the desired polypeptide and DNA encoding DHFR, but not the genome of taurine transporter. The method of producing a desired polypeptide comprises culturing the above mentioned cell and isolating the desired peptide.
EFFECT: invention enables to produce the desired polypeptide with a high yield.
9 cl, 10 dwg, 6 ex
SUBSTANCE: proposed enzymatically inactive IgA1 protease with replacement Ser267Ala for use as a component of a polyvalent vaccine designed to protect people against meningococcal infection and other microorganisms, which pathogenicity is caused by IgA1 protease. The invention includes a polynucleotide encoding the said mutant form of IgA1 protease of Neisseria meningitidis of serogroup B, comprising the said polynucleotide, a recombinant plasmid DNA, the strain Escherichia coli - producer of a mutant form of IgA1 protease according to the invention, the method of preparing a recombinant form of the enzyme using a technology of recombinant DNA, and recombinant inactivated IgA1 protease.
EFFECT: increased level of immunogenicity.
6 cl, 1 tbl, 5 dwg, 7 ex
SUBSTANCE: versions of modified yeast cells are presented. Each of the versions produces hydrogen sulfide at a reduced level and includes exogenous polynucleotide encoding a polypeptide MET10 which catalyses the transformation reaction of sulfite to sulfide at a reduced level, where the amino acid at the position 662 of the polypeptide MET10 is Ala, Asp, Glu, Phe, Gly, His, He, Lys, Leu, Asn, Gin, Arg, Val, Trp or Tyr (SEQ ID NO: 5). The expression vector is described comprising the said exogenous polynucleotide. The culture of the said cells is described. A method of preparing the said yeast cell is proposed, including the replacement of endogenous polynucleotide encoding a sulfide-active polypeptide MET10 to polynucleotide encoding MET10 with the said replacement at the position 662 SEQ ID NO: 5, and the parent yeast cell produces hydrogen sulfide. The method of reduction of H2S level in fermentation medium is described, including the contact of the fermentation medium with the mentioned above yeast cell. The fermentation medium and the fermentation product are proposed containing the said cell or the cell culture.
EFFECT: invention enables to reduce the level of H2S in the fermentation medium in production of fermented beverages.
56 cl, 8 dwg, 11 tbl, 4 ex
SUBSTANCE: there are presented an expression system for high gene expression including a promoter, and at least one MAR sequence; an isolated and purified nucleic acid molecule representing a MAR sequence; a cell; a transgenic animal; a kit for high expression, as well as the use of said expression system for increased protein production.
EFFECT: invention may be used for producing a wide range of proteins including antibodies and interferons by transgenic animals and cell cultures.
7 cl, 15 dwg, 5 tbl, 1 ex
SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.
EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.
12 cl, 3 dwg, 2 tbl, 4 ex
SUBSTANCE: what is offered is a method for inhibition of malignant tumour metastasis and tumour cell migration by reducing a KRS cell level. That is ensured by using an expression vector comprising a promoter and a functionally bound polynucleotide coding antisemantic RNA or siRNA of KRS polynucleotide, or the KRS antibody.
EFFECT: invention provides effective inhibition of malignant tumour metastasis by inhibition of the KRS function responsible for intensified tumour cell migration and observed with KRS translocation into a membrane and reaction with a laminin receptor.
8 cl, 33 dwg, 1 tbl
SUBSTANCE: method involves transformation of a Clostridium acetobutylicum cell by a vector containing: a replicon origin enabling its replication in C. acetobutylicum; a replacement cartridge containing a first marker gene surrounded by two sequences homologous to selected sites around the DNA target sequence, enabling recombination of said cartridge; a second marker gene representing upp counter-selection marker. The cells expressing the first marker gene are selected with the cartridge integrated in their genome. The cells not expressing the second marker gene with the eliminated said vector are selected.
EFFECT: invention enables producing the transformed Clostridium acetobutylicum cell which is genetically stable and marker-free.
31 cl, 6 dwg, 4 ex
SUBSTANCE: method for plasmid DNA condensation provides mixing plasmid DNA with a condensing agent in the molar ratio of 1:10. A condensing agent is presented by recombinant DNA-binding Hu-protein Acholeplasma laidlawii. The invention may be used in research studies and for the purpose of gene-therapy agent delivery into the eukaryotic cells.
EFFECT: method provides higher effectiveness of eukaryotic cell transfection by plasmid DNA.
SUBSTANCE: disclosed is a rose whose colour changes to blue, containing a flavone synthase gene of the family Scrophulariaceae, a pansy flavonoid-3′,5′-hydroxylase gene (Viola x wittrockiana), and an anthocyanin methyltransferase gene whose amino acid sequence are given in the description, and where the rose contains: (a) flavone produced by expression of the flavone synthase gene, (b) delphinidin produced by the flavonoid-3′,5′-hydroxylase gene, and (c) malvidin produced by the anthocyanin methyltransferase gene. Described is a part, tissue, a vegetative body and a cell of the rose having the same genes. Disclosed is a method of changing the flower colour to blue, involving introduction into the rose of the flavone synthase of the family Scrophulariaceae, a pansy flavonoid-3′,5′-hydroxylase gene (Viola x wittrockiana) and an anthocyanin methyltransferase gene, and involving expression of flavone synthase, flavonoid-3′,5′-hydroxylase and anthocyanin methyltransferase genes.
EFFECT: invention enables to change the colour of petals of a rose towards blue.
7 cl, 12 tbl, 13 ex
SUBSTANCE: invention involves a highly effective midkine aptamer and a pharmaceutical agent containing it.
EFFECT: aptamer enables inhibiting midkine activity and treating the related diseases.
10 cl, 12 dwg, 5 tbl, 8 ex
SUBSTANCE: culture fluid obtained after growth of the mixture of strains Lactobacillus acidophilus NKI, 100ash, K3"Ш"24, is frozen, the fat layer is removed, the defatted part is thawed and the cells are separated by centrifugation. The obtained supernatant is sterilised by membrane vacuum filtration, is concentrated with membrane ultrafiltration in the process of centrifugation, the concentrate of supernatant is treated with the components more than 30 KDa with fivefold excess of ice acetone. The resulting precipitate is diluted with 20 mM NaCl at a ratio of 2:1 by volume and is mixed with 10% Tween-80 to a final concentration of 1% Tween. Purification and isolation of the target product is carried by the method of horizontal isoelectrophoresis in a plate of 5% polyacrylamide gel with 7-8 M urea and 5% sucrose in gradient pH 4-8 at a temperature 8-12°C for 8 hours When isolation of the target product after electrophoresis the gel stripes are cut on isopoints, frozen at a temperature (-20°C) for 2 hours, thawed and dispersed, the solid parts are separated by centrifugation, concentrated by centrifugation at 5500 rev/min for 60 min at room temperature, low molecular weight impurities are removed, and the concentrate is mixed with phosphate buffer in 100-fold volume The gel stripes are cut on isopoints with pl (4.0-4.3) with the isolation of acid lectins and pl (7.6-8.0) with the isolation of alkaline pectins.
EFFECT: obtaining of lectins having anti-candidal activity is provided
5 cl, 1 tbl