WO2024016658A1 - Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法 - Google Patents

Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法 Download PDF

Info

Publication number
WO2024016658A1
WO2024016658A1 PCT/CN2023/077574 CN2023077574W WO2024016658A1 WO 2024016658 A1 WO2024016658 A1 WO 2024016658A1 CN 2023077574 W CN2023077574 W CN 2023077574W WO 2024016658 A1 WO2024016658 A1 WO 2024016658A1
Authority
WO
WIPO (PCT)
Prior art keywords
variant
pal
ammonia lyase
phenylalanine ammonia
amino acid
Prior art date
Application number
PCT/CN2023/077574
Other languages
English (en)
French (fr)
Inventor
袁哲凡
郑华宝
魏冉
唐扬洋
金城东
Original Assignee
浙江泽科塔生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江泽科塔生物医药有限公司 filed Critical 浙江泽科塔生物医药有限公司
Publication of WO2024016658A1 publication Critical patent/WO2024016658A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01005—Phenylalanine ammonia-lyase (4.3.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y403/00—Carbon-nitrogen lyases (4.3)
    • C12Y403/01—Ammonia-lyases (4.3.1)
    • C12Y403/01024—Phenylalanine ammonia-lyase (4.3.1.24)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention provides a variant of phenylalanine ammonia lyase (PAL) derived from Anabaena variabilis, including conjugates and pharmaceutical compositions of the variant, and polynucleotides for encoding the variant. , bacteria comprising the variant and methods for preparing the variant.
  • PAL phenylalanine ammonia lyase
  • Phenylalanine ammonia lyase together with histidine ammonia lyase (HAL) and tyrosine ammonia lyase (TAL), belong to the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3 )a member of. More specifically, enzymes with PAL activity (EC 4.3.1.23-1.25 and previously classified as EC 4.3.1.5) catalyze the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid.
  • PAL is a non-mammalian enzyme that is widely distributed in plants, fungi, and a limited number of bacteria.
  • the PAL enzyme can be used as a therapeutic protein to treat the metabolic disorder phenylketonuria (PKU).
  • PKU is an autosomal genetic disorder of metabolism in which the liver enzyme phenylalanine hydroxylase (PAH), or one or more enzymes involved in the synthesis or recycling of the cofactor tetrahydrobiopterin, occurs in the corresponding gene due to Mutations resulting in lack of function. A lack of this function results in higher levels of phenylalanine in the bloodstream. Higher phenylalanine is converted into phenylpyruvate (phenyl ketone) and other derivatives.
  • PAH liver enzyme phenylalanine hydroxylase
  • PEG-PAL injectable recombinant PAL and PAL variants modified by pegylation
  • Pegylation methods have been shown to improve enzyme half-life and reduce subject antigen responses (see WO 2008/153776, WO 2011/097335 and US7531341).
  • PAL variants that are effective in PEG-PAL compositions have been described as wild-type Nostoc punctiforme (NpPAL); Anabaena variabilis (PAL) and Rhodosporidium toruloides )(RtPAL) variant.
  • variants of wild-type PAL have been described in which the cysteine residues at positions 64, 318, 503 and 565 are replaced by serine (see US7790433; US7560263 and US7537923).
  • PAL potentially has various therapeutic uses, factors such as reduced specific activity and proteolytic instability limit the use of PAL.
  • factors such as reduced specific activity and proteolytic instability limit the use of PAL.
  • the use of PAL as enzymatic therapy is associated with several drawbacks, such as immunogenicity and proteolytic susceptibility (see Vellard, Curr. Opin. Biotechnol. 14:1-7 (2003)).
  • PAL a delicate balance between substrate affinity and enzyme activity is required to achieve and maintain control of plasma phenylalanine levels at normal but somewhat narrow levels in diseases characterized by hyperphenylalaninemia. In the range. To date, no concerted efforts aimed at improving these parameters have been made due to the lack of structural and biochemical knowledge about this protein.
  • HPA hyperphenylalaninemia
  • other diseases such as cancer
  • the object of the present invention is to provide a phenylalanine ammonia lyase (PAL) variant according to the invention or obtained by a method according to the invention, the variant having SEQ ID NO: 1
  • a variant of the amino acid sequence of Anabaena phenylalanine ammonia lyase (UniProtKB/Swiss Prot:Q3M5Z3.1), or the variant has SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 The amino acid sequence shown.
  • PAL variants according to the present invention may include one or more amino acid substitutions or combinations of amino acid substitutions, for example, including one or a combination of more than two of the following: C503S/C565L, C503S/C565P, C503L/C565P mutations, and T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G218N, M222L, M222I, N437S, S438N, S438A, I439V, I439C, I439A, I439K, A440T, A440G, A440V, A440 T, R441N, R442Q, T445V, T445S, A447S, E448K, N453G, Q457L and G458K.
  • the PAL variant enzyme according to the invention has phenylalanine ammonia lyase activity.
  • the PAL variant enzymes according to the invention or obtained by the method according to the invention can be used to treat diseases or disorders characterized by elevated blood concentrations of phenylalanine.
  • the PAL variant enzymes according to the invention can be used to treat phenylketonuria (PKU).
  • the PAL variant enzyme according to the invention has enhanced catalytic activity from phenylalanine to trans-cinnamic acid, enhanced thermal stability and low aggregation.
  • the terms “comprises” and “includes” are to be construed as open-ended, allowing for the inclusion of additional elements in addition to those enumerated.
  • the terms “consisting of” and “consisting of” shall be construed as closed and shall not include parts other than those listed in the claims.
  • the term “consisting essentially of” shall be construed as partially closed, i.e., only additional ingredients that do not fundamentally alter the claimed properties of the subject matter are permitted.
  • the modal verb "may” or “might” refers to the preferred use or selection of one or more options or choices among the several embodiments or features described. If no options or choices are disclosed regarding a particular embodiment or feature included therein, the modal verb "may” refers to an affirmative action as to how the described embodiment or feature may be performed or used.
  • the modal verb "can” has the same meaning and connotation as the auxiliary verb "can”.
  • Cysteinyl Cys(C) Glutamine: Gln(Q)
  • Threonine Thr(T) Tryptophan: Trp(W)
  • amino acid residue includes, but is not limited to, alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), benzene Alanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), lysine (Lys or K), leucine (Leu or Amino acid residues contained in the group consisting of L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or
  • protein protein covalently linked to an amide bond through an amide bond, independent of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • an “analog” or “derivative” as used in the context of the present invention is a compound that has greater than about 70% but less than 100% sequence similarity to a given compound, such as a peptide. These analogs or derivatives may contain non-naturally occurring amino acid residues as well as naturally occurring amino acid residues. These analogs or derivatives may also contain one or more D-amino acid residues, and may contain non-peptide linkages between two or more amino acid residues.
  • polynucleotide refers to a polymer composed of nucleotide units.
  • Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs.
  • Nucleic acid analogs include non-naturally occurring bases, nucleotides that participate in bonds to other nucleotides other than naturally occurring phosphodiester bonds, or include bases linked by linkages other than phosphodiester bonds. base.
  • nucleotide analogs include, for example, but not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, borane phosphates, methyl phosphonates, chiral methyl phosphonates, 2-O-methylribonucleotide, peptide-nucleic acid (PNA), etc.
  • PNA peptide-nucleic acid
  • nucleotide analogs include, for example, but not limited to, phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, borane phosphates, methyl phosphonates, chiral methyl phosphonates, 2-O-methylribonucleotide, peptide-nucleic acid (PNA), etc.
  • PNA peptide-nucleic acid
  • nucleic acid generally refers to large polynucleotides.
  • oligonucleotide generally refers
  • nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C)
  • this also includes an RNA sequence (i.e., A, U, G, C), where "U” is substituted for "T” .
  • a vector such as an expression vector, containing a nucleic acid encoding one or more polypeptides and/or proteins according to the invention.
  • the term "encoding" refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA or mRNA that serves as a template for the synthesis of other polymers and macromolecules used in biological processes.
  • Objects and macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA produced by a gene in a cell or other biological system produces a protein, the gene encodes that protein.
  • Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing) and the non-coding strand used as a template for transcription of a gene or cDNA can be said to encode the protein or other product of the gene or cDNA.
  • a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are in degenerate form of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can only contain introns.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked
  • expression vector refers to a vector comprising a recombinant polynucleotide containing the same nucleotide as the nucleotide to be expressed. Sequence is operable linked expression control sequences.
  • the expression vector contains sufficient cis-acting elements for expression; other expression elements can be provided by the host cell or in vitro expression system.
  • Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (eg, naked or contained in liposomes) and viruses incorporating recombinant polynucleotides.
  • recombinant polynucleotide refers to a polynucleotide having sequences that are not linked together in nature.
  • the amplified or assembled recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform a suitable host cell.
  • a host cell containing a recombinant polynucleotide is called a "recombinant host cell.”
  • the gene is then expressed in a recombinant host cell to produce, for example, a "recombinant polypeptide.”
  • Recombinant polynucleotides can also serve non-coding functions (e.g., promoters, origins of replication, ribosome binding sites, etc.).
  • the expression vector is a "plasmid" which refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated.
  • insertion refers to the addition of one or more nucleotides to a native polynucleotide sequence.
  • the engineered strains disclosed herein may include the insertion of one or more genes.
  • the engineered strains disclosed herein include insertion of a sequence encoding an exogenous phenylalanine ammonia lyase (PAL).
  • the engineered strains disclosed herein include insertions in endogenous genes (i.e., genomic insertions) that result in non-functional gene products.
  • substitution refers to the replacement of a nucleotide of a native polynucleotide sequence with a nucleotide not native to the polynucleotide sequence.
  • the engineered strains disclosed herein may include substitutions in one or more genes. In some embodiments, the substitution results in a non-functional gene product, for example, wherein the substitution introduces a premature stop codon (eg, TAA, TAG, or TGA) in the coding sequence of the gene product. In some embodiments according to the invention, engineered strains according to the invention may include two or more substitutions, wherein the substitutions introduce multiple premature stop codons (eg, TAATAA, TAGTAG, or TGA).
  • the term "primer” refers to a polynucleotide capable of specifically hybridizing to a specified polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that induce synthesis, ie, in the presence of a nucleotide, a complementary polynucleotide template, and a polymerization reagent such as a DNA polymerase. Primers are generally single-stranded, but can also be double-stranded. Primers are typically DNA, but many types of synthetic and naturally occurring primers are also available for many applications.
  • the primer is complementary to the template to which it is designed to hybridize to serve as a site for initiating synthesis, but does not need to reflect the exact sequence of the template.
  • the specific hybridization of the primer to the template depends on the stringency of the hybridization conditions.
  • Primers can be labeled with, for example, a chromogenic, radioactive or fluorescent moiety and used as a detectable moiety.
  • wild type refers to the natural genetic form of an organism. Wild type is different from mutant forms (organisms with genetic mutations).
  • variant according to the invention PAL variant
  • variant of phenylalanine ammonia lyase PAL
  • variant enzyme PAL enzyme
  • PAL enzyme PAL enzyme
  • the term "effective amount” refers to a dose sufficient to produce the desired results on the health conditions, pathologies and diseases of an individual or for diagnostic purposes. Desired outcomes may include subjective or objective improvement in dose recipients.
  • a “therapeutically effective amount” refers to an amount of a drug effective to produce the desired beneficial effect on health. In any individual case, the appropriate “effective” amount can be determined by one skilled in the art using routine experimentation.
  • treatment means prophylactic treatment or therapeutic treatment or diagnostic treatment.
  • the term "pharmaceutical composition” refers to a composition suitable for use as a medicament in an animal subject, including humans and mammals.
  • the pharmaceutical composition contains a pharmacologically effective amount of PAL polypeptide and a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions include active ingredients and inert ingredients constituting carriers, as well as combinations, complexes or aggregations of any two or more ingredients, or dissociations of one or more ingredients, or other combinations of one or more ingredients. Any product resulting directly or indirectly from a type of reaction or interaction. Accordingly, pharmaceutical compositions of the invention include any composition made by mixing a compound or conjugate of the invention with a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to standard pharmaceutical carriers, buffers and excipients (e.g. phosphate buffer, 5% dextrose in water) and emulsions (e.g. water-in-oil or water-in-water). oil emulsions) and any of various types of wetting agents and/or adjuvants.
  • a PAL variant, PAL variant enzyme, PAL mutant or analogue according to the invention.
  • the PAL is derived from Anabaena variabilis and has the amino acid sequence shown in SEQ: ID NO: 1
  • the PAL variant according to the present invention has any one or two or more selected from the following Combinations of amino acid substitutions: C503S/C565L, C503S/C565P, C503L/C565P, T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G218N, M222L, M222I, N437S, S438N, S43 8A, I439V, I439C, I439A, I439K, A440T, A440G, A440V, A440T, R441N, R442Q, T445V, T445S, A447S, E
  • the PAL variant enzyme according to the invention has phenylalanine ammonia lyase activity (EC 4.3.1.24).
  • the PAL variant enzyme according to the invention has enhanced phenylalanine ammonia lyase activity (EC 4.3.1.24) relative to the wild-type PAL enzyme.
  • the PAL variant according to the invention comprises the amino acid substitution C503L/C565P/222L, or the amino acid substitution C503L/C565P/G218A.
  • the activity of the variant is higher than that of the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO:1 .
  • the variant exhibits the same stability at a temperature ranging from 37°C to 70°C as compared with the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO:1 or higher stability, in other words, the PAL variant according to the invention has the same or higher, and preferably higher stability than wild-type PAL at temperatures in the range of 37°C to 70°C.
  • the variant has low aggregation compared with the phenylalanine ammonia lyase comprising the amino acid sequence shown in SEQ ID NO: 1.
  • the PAL variant or derivative according to the invention may have an amino acid sequence that includes conservative amino acid substitutions or non-conservative amino acid substitutions relative to the reference amino acid sequence SEQ: ID NO: 1.
  • a PAL variant or derivative peptide, polypeptide or protein according to the invention may include conservative amino acid substitutions and/or non-conservative amino acid substitutions relative to a reference peptide, polypeptide or protein.
  • Consservative amino acid substitutions refer to substitutions that are expected to have minimal interference with the properties of the reference peptide, polypeptide or protein.
  • each of the following six groups contains amino acids that are conservative substitutions for each other: (1) Alanine (A) , serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamic acid (Q); (4) ) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) benzene Alanine (F), tyrosine (Y), tryptophan (W).
  • “Non-conservative amino acid substitutions” are those substitutions that are expected to interfere most with the properties of the reference peptide, polypeptide, or protein. In other words, conservative amino acid substitutions substantially preserve the structure and function of the reference peptide, polypeptide, or protein, whereas non-conservative amino acid substitutions do not preserve the structure and function of the reference peptide, polypeptide, or protein.
  • PAL variants according to the invention can be expressed in plants, fungi and bacteria.
  • the PAL variant according to the present invention can be expressed in Nodida punctata, Anabaena variabilis, Rhodosporium toruloides, Streptomyces marinum, Indocystis nidulans, Photorhabditis luminescens, Streptomyces versicolor, Escherichia coli.
  • the PAL variant according to the present invention can be expressed in probiotics or engineered bacteria that are capable of polynucleotide recombination and are not pathogenic bacteria and do not produce toxins.
  • the PAL variant according to the present invention can be produced by introducing the recombinant polynucleotide encoding the PAL variant according to the present invention into the probiotic or engineered bacteria and allowing it to be expressed.
  • the probiotic or engineered bacteria include E. coli and G + bacteria such as Bacillus subtilis.
  • the invention provides a conjugate comprising a PAL variant according to the invention, wherein the conjugate comprises a PAL variant according to the invention and a suitable polymer, and in particular consists of a PAL variant according to the invention.
  • the inventive PAL variants are composed of suitable water-soluble polymers.
  • the suitable water-soluble polymers include polyethylene glycol, polyzwitterionic polymers, and polyoxazolines.
  • the water-soluble polymer is polyethylene glycol.
  • the invention provides a pharmaceutical composition comprising: (i) a PAL variant enzyme according to the invention; and (ii) a suitable pharmaceutically acceptable carrier.
  • a pharmaceutical composition according to the invention includes: (i) a conjugate of a PAL variant enzyme according to the invention; and (ii) a suitable pharmaceutically acceptable carrier. It is contemplated that the conjugate of a PAL variant enzyme comprises a PAL variant enzyme chemically coupled to a polyethylene glycol (PEG) polymer.
  • PEG polyethylene glycol
  • the PAL variant in the pharmaceutical composition according to the invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • a pharmaceutical composition according to the invention is formulated and administered to a subject in need thereof.
  • the pharmaceutical compositions according to the present invention may be formulated and/or administered according to dosages and techniques well known to those skilled in the medical field, taking into account factors such as the age, sex, weight and condition of a particular patient and the route of administration.
  • the pharmaceutical composition according to the invention contains pharmaceutically acceptable carriers, diluents, excipients and surfactants.
  • the pharmaceutical composition may also include a preservative (eg, an antibacterial agent or an antibacterial agent such as benzalkonium chloride).
  • the pharmaceutical composition may further comprise a buffering agent (for example, in order to maintain the pH of the composition between 6.5 and 7.5).
  • the pharmaceutical composition according to the invention can be used to treat phenylketonuria.
  • the invention provides a polynucleotide encoding a PAL variant according to the invention, the polynucleotide being DNA or RNA, in particular the polynucleotide being cDNA or mRNA or tRNA.
  • DNA and RNA have specific nucleotide sequences so that the PAL variant enzyme according to the present invention can be synthesized using them as templates.
  • the polynucleotide according to the invention is codon-optimized such that said polynucleotide can be used to express a PAL variant according to the invention in E. coli.
  • the polynucleotide according to the invention is codon-optimized such that said polynucleotide can be used to express a PAL variant according to the invention in animal cells, in particular human cells.
  • the polynucleotide according to the invention is codon optimized, enabling expression of the PAL variant enzyme according to the invention in E. coli.
  • the polynucleotide according to the invention is codon optimized, enabling expression of the PAL variant enzyme according to the invention in human cells.
  • the disclosed polynucleotides may be present in vectors known in the art (eg, plasmid vectors).
  • the base "GC" that is, the first methionine, is introduced after the start codon of the enzyme cleavage site NcoI.
  • a glycine is introduced later to prevent frameshifting during translation.
  • the PAL variant for example, a variant of Anabaena phenylalanine ammonia lyase PAL (SEQ ID NO: 1), for example, includes Other PAL variants according to the present invention are PAL variants of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
  • an expression vector according to the present invention comprising the polynucleotide according to the present invention, wherein the expression vector can be expressed in living cells.
  • expression vectors according to the invention include all expression vectors known in the art, such as cosmids, plasmids (eg, naked or contained in liposomes) and integrated recombinant polynucleotides of viruses.
  • the expression vector according to the invention is a viral vector, such as adenovirus, adeno-associated virus and lentivirus.
  • the expression vector according to the invention is a plasmid
  • the plasmid contains a promoter
  • the promoter is operably linked to the polynucleotide according to the invention.
  • the plasmid enters the cell by transfection with liposomes or cationic polymers.
  • the invention provides a bacterium according to the invention, wherein the bacterium has a polynucleotide according to the invention integrated into its genome and/or is capable of expressing a PAL variant according to the invention.
  • the invention also provides a modified cell according to the invention, said modified cell comprising a PAL variant according to the invention and/or a polynucleotide according to the invention and/or an expression vector according to the invention, wherein any Optionally, the modified cells are animal cells, especially human cells.
  • a host cell eg a bacterium according to the invention or a modified cell according to the invention
  • said polynucleotide is operably Linked to one or more control sequences for expression of the PAL variant in the host cell.
  • Suitable host cells for expressing PAL variants according to the invention are, for example, bacteria according to the invention, such as E.
  • coli Vibrio fluvialis, Streptomyces and Salmonellatyphimurium cells
  • fungal cells Such as yeast cells (eg, Saccharomyces cerevisiae or Pichia pastoris); insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, BHK, 293, and Bowes Melanoma cells; and plant cells.
  • yeast cells eg, Saccharomyces cerevisiae or Pichia pastoris
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, BHK, 293, and Bowes Melanoma cells
  • plant cells such as CHO, COS, BHK, 293, and Bowes Melanoma cells
  • Exemplary host cells also include various Escherichiacoli strains (eg, W3110 ( ⁇ fhuA) and BL21 (DE3))
  • the invention provides a method for preparing a PAL variant according to the invention, said method comprising culturing a bacterium according to the invention or a modified cell according to the invention in a culture medium to produce a method according to the invention. and isolating the PAL variant according to the invention from said modified cells and/or said culture medium.
  • the PAL variant enzymes obtained by the method according to the invention can be used to treat diseases or disorders characterized by elevated blood concentrations of phenylalanine, such as phenylketonuria (PKU).
  • PKU phenylketonuria
  • the PAL variant enzyme obtained by the method according to the present invention has one or more characteristics selected from the following compared to the PAL enzyme derived from Anabaena variabilis (SEQ ID NO: 1) Amino acid substitutions: C503S/C565L, C503S/C565P, C503L/C565P mutations and T102R, T102H, I165L, I165V, G166A, G166S, G218A, G218S, G218N, M222L, M222I, N437S, S438N, S438A ,I439V,I439C,I439A, I439K, A440T, A440G, A440V, A440T, R441N, R442Q, T445V, T445S, A447S, E448K, N453G, Q457L and G458K.
  • SEQ ID NO: 1 Amino acid substitutions: C503S/C565L
  • the method according to the present invention specifically includes one or more of the following steps: (i) constructing a PAL original enzyme expression system including plasmids, expression cells or the like; (ii) from a PAL host Extract the pET28a-PAL plasmid from cells; (iii) use the plasmid obtained from step (ii) as a template to construct a mutation library using site-directed mutagenesis technology; (iv) use a high-throughput screening method to cultivate PAL variants and detect mutations Screening for optimal mutations using somatic enzyme activity.
  • the components in the context of the present invention may be crude and/or at least partially isolated and/or purified.
  • isolated or purified may refer to a component removed from its natural environment and/or medium and at least 60% free, preferably at least 75% free, and more preferably at least 90% free, even more Preferably they are at least 95% free from other components and/or media with which they are naturally associated.
  • PAL enzyme variants suitable for use in the method include, but are not limited to, variants of the PAL enzyme derived from Anabaena variabilis.
  • Suitable transformed cells may include, but are not limited to, transformed E. coli, wherein the PAL gene sequence has preferably been codon optimized for expression of PAL enzyme variants according to the invention in E. coli.
  • the culture medium can be further supplemented and/or modified to promote the growth of the transformed cells.
  • glucose is added to the culture medium at an optional concentration of 0.1-0.3% (v/v).
  • transformed cells can be passaged one or more times. For example, after the culture medium reaches an OD600 of at least about 1-2 (e.g., while the transformed cells are still in the growth phase), the transformed cells can be removed from the culture medium and placed in a solution containing phenylalanine. in fresh Minimum Essential Medium. This optional subculture step can be performed one or more times.
  • a subculture step is performed to remove transformed cells from the culture when the culture contains an excess of tCA, for example, when the culture medium contains at least 0.5, 1.0, 1.5 or 2.0mM tCA.
  • the invention provides a method of treating a disease or condition characterized by elevated levels of phenylalanine in a subject in need thereof, wherein said method Comprising: administering to the subject the PAL variant according to the first aspect of the present invention; the conjugate according to the second aspect of the present invention; a third party according to the present invention
  • the administration is by any suitable method known in the art, e.g., oral, transdermal, transmucosal , intrapulmonary (including aerosolization), intramuscular, subcutaneous or intravenous administration.
  • administration is by subcutaneous or oral administration.
  • the dosage form is selected for oral or parenteral formulation in a manner known in the art.
  • Pharmaceutical compositions according to the invention may be, for example, in the form of solid, semi-solid or liquid dosage forms, such as tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions or the like, For example, unit dosage forms suitable for single administration of precise doses.
  • the pharmaceutical composition according to the present invention may include a therapeutically effective amount of the PAL variant according to the present invention combined with a pharmaceutically acceptable carrier; in addition, other pharmaceutical agents, adjuvants, diluents, buffers, etc. may optionally be included .
  • a slow release or sustained release system may be used in order to maintain a constant dosage level.
  • the PAL variant according to the first aspect of the present invention or the conjugate according to the second aspect of the present invention is administered subcutaneously to the subject to treat phenylalanine-based Diseases and conditions characterized by elevated levels, in particular phenylketonuria (PKU).
  • PKU phenylketonuria
  • the subject is administered a conjugate according to the second aspect of the invention subcutaneously, thereby increasing the half-life of the PAL variant enzyme when administered to a subject in need thereof.
  • the conjugate comprises a PAL variant enzyme chemically coupled to a polyethylene glycol (PEG) polymer.
  • Figure 1 shows a schematic diagram of phenylalanine ammonia lyase (PAL) or a PAL variant catalyzing phenylalanine to generate trans-cinnamic acid;
  • PAL phenylalanine ammonia lyase
  • Figure 2A schematically shows the three-dimensional structure of a single subunit in the PAL tetramer
  • Figure 2B schematically shows the position of some mutation sites on the three-dimensional structure
  • Figures 3A and 3B schematically illustrate PAL high-throughput screening sites and amino acid substitutions with phenylalanine ammonia lyase activity according to the present invention
  • FIG 4 schematically shows the SEC-HPLC chromatogram (SEC chromatography column Ultrahydrogel 1000 ( Waters), mobile phase: 0.3ml/min PBS buffer solution);
  • Figure 5 schematically shows the SDS-PAGE diagram of some PAL variants purified by affinity chromatography according to the present invention
  • Figure 6 schematically shows the changes in enzyme activity of the PAL variant C503L/C565P according to the present invention at a pH value between 6.5-9.0;
  • Figure 7 schematically shows the thermal stability changes of the enzymatic activity of PAL variants according to the present invention and wild-type PAL at a temperature of 37-70°C;
  • Figure 8 schematically shows the HPLC-SEC patterns of some PAL variants before and after modification with polyethylene glycol (PEG) according to the present invention.
  • Codon optimization was performed on the phenylalanine ammonia lyase PAL encoding gene derived from Anabaena variabilis (the amino acid sequence is shown in SEQ ID NO.1), and the nucleotide sequence of the codon-optimized PAL gene is shown in SEQ ID NO:2 shown.
  • the codon-optimized nucleotide sequence of the PAL gene (SEQ ID NO. 2) was artificially synthesized and inserted between Nco I and Xho I of pET28a to obtain the recombinant plasmid pET28a-PAL.
  • the electrophoresis diagram is shown in Figure 2.
  • composition of LB liquid culture medium is: yeast extract 5g/L, tryptone 10g/L, NaCl 10g/L, the solvent is distilled water, and the pH is about 7.0.
  • Example 2 Using the plasmid pET28a-PAL prepared in Example 1 as a template, design a pair of primers, and set the mutation site primer sequence to "NNN”. Use inverse PCR technology to amplify the entire plasmid, add DpnI to digest the plasmid, and use the kit ( TaKaRa) purified plasmid, and introduced the plasmid into the cloning host strain DH5 ⁇ using the electrotransformation method. After the plasmid was enriched by the cloning bacteria, the plasmid was extracted, and then the plasmid was transformed into E. coli BL21 (DE3). The primers for each point saturation mutation are shown in Table 1.
  • the reverse PCR amplification system is shown in Table 2.
  • the PCR reaction process is as follows, and the PCR product is detected by 0.8% agarose gel electrophoresis.
  • the supernatant liquid is the crude enzyme liquid.
  • enzyme activity unit The deamination of 1 ⁇ mole L-phenylalanine into trans-cinnamic acid and NH 3 per minute at 37°C and pH 8.5 is defined as 1 U.
  • the enzyme activity calculation formula is as follows:
  • ⁇ c product concentration change, unit uM, by establishing a linear relationship between product concentration and absorbance, substitute into the detected product concentration change;
  • V1 The total volume of the activity measurement system, the unit is ml;
  • V2 volume of enzyme solution added, unit is ml
  • Example 4 Cell culture and protein induction of pET28a-PAL and its mutant plasmid expression bacteria
  • Protein concentration detection refers to the (Thermo) method of the BCA detection kit, which is incorporated into this article by reference; the linear range is from 25ug/ml to 2000ug/ml. This implementation case uses a 96-well detection plate, and the added volumes of enzyme solution and detection solution are 10ul and 200ul, or 25ul and 200ul respectively.
  • Enzyme activity detection The enzyme activity detection method described in Example 3 was used to detect the enzyme activity of wild-type PAL and PAL variants.
  • Cell lysis Thaw the frozen cell pellets and resuspend them in a buffer of 20mM Tris-HCl and 100mM NaCl with a pH value of 8.0 at room temperature to form a cell slurry with a density of approximately 120 to 140OD600.
  • the cells were passed twice through a Niro NS30006 homogenizer at 700-800 Bar (where the temperature was controlled below 30°C) to lyse the cells by homogenization. After homogenization, the pH was adjusted to approximately 8.0 by adding 1N NaOH to the lysate. Gradually heat the cell lysate to 55°C, maintain 55°C for 30 to 120 min, and then cool. Centrifuge to clarify the cell lysate.
  • FIG. 5 shows a total of 9 lanes, in which from left to right are the protein standard Marker (lane 1) and the PAL variant containing the His tag (lane 2 to lane 1). Lane 9), where lanes 2 to 9 represent in order: PAL variants containing C503L/C565P mutations, PAL variants containing mutations T102R/C503L/C565P, PAL variants containing G218A/C503L/C565P mutations, PAL variants containing G218S/C503L/C565P mutations, PAL variants containing M222L/C503L/C565P mutations, PAL variants containing S438N/C503L/C565P mutations, and PAL variants containing I439V/C503L/C565P mutations; wherein: The resulting standard strips from top to bottom are 200, 140, 95, 65, 52, 41, 33
  • the Tris resuspended enzyme solution is passed through a hydrophobic interaction (HIC) column and an anion exchange (AIEX) column in sequence to purify the PAL variant.
  • HIC hydrophobic interaction
  • AIEX anion exchange
  • HIC chromatography column (t-Butyl, Tosoh) was pre-equilibrated with Tris-HCl buffer solution (50mM, pH 8.5) containing 1M (NH 4 ) 2 SO 4 .
  • the PAL enzyme solution was diluted 2 times with Tris-HCl buffer solution (50mM, pH 8.5) containing 2M (NH 4 ) 2 SO 4 , and loaded into the above HIC chromatography column, and the solution was filled with 1M to 0M (NH 4 ) 2 Gradient elution with Tris-HCl buffer solution of SO 4 (50mM, pH 8.5), collecting the components with enzymatic activity, and performing ultrafiltration, concentration and desalting.
  • Example 7 Comparison of aggregation properties of wild-type PAL enzyme and PAL variant enzyme
  • the purified PAL was concentrated, and the concentrated protein solution (2.5 mg/ml) was incubated at 37°C for 2 hours to accelerate the aggregation of the purified PAL protein in the solution.
  • PAL protein was separated by SEC-HPLC to detect aggregation. to confirm If disulfide cross-linking is the cause of aggregation, 50 mM dithiothreitol (DTT) is added to the concentrated protein solution and then incubated at 37°C for 2 hours.
  • DTT dithiothreitol
  • the solution was further concentrated to approximately 10 mg/mL by ultrafiltration and incubated at 37°C for 2 hours.
  • the solution was further concentrated to approximately 30 mg/mL and incubated at 37°C for 2 hours.
  • the purified wild-type PAL enzyme aggregated after incubation at 37°C for 2 hours. As expected, this aggregation was exacerbated when PAL protein was concentrated prior to incubation at 37°C for 2 h. Exposure of concentrated proteins to DTT prevented aggregation, indicating that aggregation was caused by disulfide cross-linking. In contrast, the purified PAL variant (C503S/C565P) and PAL variant (C503S/C565L), etc. did not form aggregates after incubation at 37°C for 2 hours, indicating that the PAL variant is more comparable to the wild-type PAL enzyme. Has lower aggregation.
  • Example 8 pH adaptation curves of wild-type PAL enzyme and PAL variant enzyme
  • the optimal pH values for wild-type PAL enzyme and selected PAL variants were determined by specific activity assays as described in Example 5. Take 10ul of pure enzyme solution (total protein 0.2 ⁇ g) + 190ul of 100mM Tris-HCl (pH8.5) buffer dissolved in 22.5mM Phe for measurement, and perform enzyme activity detection in a 200 ⁇ L system at 37°C.
  • the 100mM Tris-HCl (pH8.5) buffer dissolved in 22.5mM Phe was set to pH values of 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 respectively.
  • Example 9 Temperature stability of wild-type PAL enzyme and PAL variant enzyme
  • the effect of temperature on the stability of wild-type PAL enzyme and selected PAL variant enzymes was determined by incubating the protein in 50mM Tris-HCl, pH 7.5, for 2 hours in the temperature range from 37°C to 70°C, and then measuring the enzyme activity. Sure. Each enzyme reaction used 1 ⁇ g PAL protein and 22.5 mM phenylalanine as substrate, and the total reaction volume was 200 ⁇ L at 37°C.
  • PEG PEG-NHS
  • the reaction solution of PEG and PAL is allowed to react at room temperature for 2-4 hours, or at 4°C overnight.
  • 10 ⁇ l of the above reaction solution is immediately diluted, and the enzyme activity is measured according to the method in Example 5, and the change in enzyme activity before and after the reaction between PAL and PEG is calculated (see Table 7).
  • the reaction solution containing the PEG-PAL conjugate was placed in a dialysis bag (molecular weight cutoff 300 kD) and fully dialyzed in PBS 7.4 buffer solution.
  • the final product was detected by HPLC-SEC.
  • the three PAL variants after being modified with PEG, the three PAL variants all showed an obvious left shift in the HPLC-SEC spectrum, indicating the successful conjugation of the PAL variants with PEG and the overall molecular size. Significant increase.
  • the steps of the methods may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Unless otherwise indicated herein or otherwise clearly contradicted by context, these steps may be repeated or repeated any number of times to achieve the intended goals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供一种苯丙氨酸解氨酶(PAL)的变体，该苯丙氨酸解氨酶(PAL)来源于变异鱼腥藻并包含SEQ ID NO:1所示的氨基酸序列，其中，所述变体包含氨基酸取代C503S/C565L，使得所述变体包含SEQ ID NO:2所示的氨基酸序列；或氨基酸取代C503S/C565P，使得所述变体包含SEQ ID NO:3所示的氨基酸序列；或氨基酸取代C503L/C565P，使得所述变体包含SEQ ID NO:4所示的氨基酸序列。本发明还提供一种包括该变体的缀合物和药物组合物、用于编码该变体的多核苷酸，包括该变体的细菌以及用于制备该变体的方法。

Description

PAL变体、包含该PAL变体的药物组合物以及用于制备该PAL变体的方法
相关申请的交叉引用
本申请要求于2022年07月18日向中国国家知识产权局提交的第202210839871.7号中国专利申请的优先权和权益，该申请的全部内容通过引用并入本文。
技术领域
本发明提供了一种来源于变异鱼腥藻的苯丙氨酸解氨酶(PAL)的变体，包括该变体的缀合物和药物组合物、用于编码该变体的多核苷酸，包括该变体的细菌以及用于制备该变体的方法。
背景技术
苯丙氨酸解氨酶(PAL)与组氨酸解氨酶(HAL)和酪氨酸解氨酶(TAL)—起属于芳香族氨基酸裂解酶家族(EC 4.3.1.23-1.25和4.3.1.3)的成员。更具体地，具有PAL活性的酶(EC 4.3.1.23-1.25和先前分类为EC4.3.1.5)催化L-苯丙氨酸的非氧化性脱氨基为反式肉桂酸。PAL非哺乳动物酶，其广泛分布于植物、真菌和有限数目的细菌中。PAL酶可以用作治疗代谢紊乱苯丙酮尿症(PKU)的治疗性蛋白。PKU是常染色体代谢遗传紊乱，其中肝酶苯丙氨酸羟化酶(PAH)或参与辅因子四氢生物蝶呤的合成或再循环的一种或更多种酶，由于在相应基因中发生突变而缺乏功能。这种功能的缺乏导致血流中苯丙氨酸水平较高。较高的苯丙氨酸被转化为苯丙酮酸盐(苯基酮)和其他衍生物。如果PKU未及时治疗，人体中的高水平的苯丙氨酸和其分解产物的部分可引起显著医学问题，所述医学问题包括智力障碍、小头畸形和癫痫发作。许多研究都集中在通过酶替代将PAL用于治疗PKU(Ambrus等人，Science 201:837-839[1978]；Bourget等人，Appl Biochem.Biotechnol，10:57-59[1984]；和Sarkissian等人，Proc.Natl.Acad.Sci.USA 96:2339-2344[1999])。
用于去除血清中苯丙氨酸的一种方法是使用可注射的重组PAL和通过聚乙二醇化修饰的PAL变体(PEG-PAL)。聚乙二醇化的方法已被证明可以改进酶的半衰期并降低受试者抗原反应(参见WO 2008/153776、W0 2011/097335和US7531341)。在PEG-PAL组合物中有效的PAL变体已经被描述为野生型点状念珠藻(Nostoc punctiforme)(NpPAL)；变异鱼腥藻(Anabaena variabilis)(PAL)和圆红冬孢酵母(Rhodosporidium toruloides)(RtPAL)的变体。 特别地，已描述了野生型PAL的变体，其中在位点64、318、503和565处的半胱氨酸残基被丝氨酸替换(参见US7790433；US7560263和US7537923)。
虽然PAL潜在地具有不同的治疗用途，但是降低的特异性活性以及蛋白水解不稳定性等因素使得PAL的用途受限。跟其他治疗性蛋白质类似，PAL用作酶疗法伴随着多个缺点，例如免疫原性和蛋白水解敏感性(见Vellard,Curr.Opin.Biotechnol.14:1-7(2003))。此外，在底物亲和力和酶活性之间需要一种微妙的平衡，以实现和维持在以高苯丙氨酸血症为特征的疾病中，将血浆苯丙氨酸水平控制在正常但有些窄的范围内。到目前为止，由于缺乏有关这种蛋白质的结构和生化知识，尚未做出以改善这些参数为目的的协调一致的努力。
因此，仍然需要具有最佳动力学特征(包含强效的催化活性、较长的生物半衰期、较大的生化稳定性和/或减弱的免疫原性)的PAL分子，以用于治疗用途，例如高苯丙氨酸血症(hyperphenylalaninemia,HPA，例如PKU)以及其他疾病(例如癌症)的治疗。
发明内容
为了解决上述问题，本发明的目的在于提供根据本发明的或通过根据本发明的方法获得的苯丙氨酸解氨酶(PAL)变体，该变体为具有SEQ ID NO:1所示的氨基酸序列的鱼腥藻苯丙氨酸解氨酶(UniProtKB/Swiss Prot:Q3M5Z3.1)的变体，或者该变体具有SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4所示的氨基酸序列。根据本发明的PAL变体可包括一个或多个氨基酸替换或氨基酸替换组合，例如包括以下中的一项或两项以上的组合：C503S/C565L，C503S/C565P，C503L/C565P突变，以及T102R，T102H，I165L，I165V，G166A，G166S，G218A，G218S，G218N，M222L，M222I，N437S，S438N，S438A，I439V，I439C，I439A，I439K，A440T，A440G，A440V，A440T，R441N，R442Q，T445V，T445S，A447S，E448K，N453G，Q457L和G458K。特别地，根据本发明的PAL变体酶具有苯丙氨酸解氨酶活性。
根据本发明的或者通过根据本发明的方法获得的PAL变体酶可以用于治疗以苯丙氨酸血浓度升高为特征的疾病或紊乱。具体而言，根据本发明的PAL变体酶可用于治疗苯丙酮尿症(PKU)。
根据本发明的一个实施方案，根据本发明的PAL变体酶具有从苯丙氨酸到反式肉桂酸的增强的催化活性、增强的热稳定性和低聚集性。
在本文使用以下以及整个申请中所述的定义来描述根据本发明的主题。
定义和术语
可以使用以下定义和术语进一步描述所公开的主题。本文中使用的定义和术语仅用于描述特定实施例的目的，并不旨在限制。在本发明上下文中，“大概”、“大约”、“实 质上”，以及“显著地”将被本领域的普通技术人员理解，并且将在一定程度上取决于其使用的上下文。如果本领域普通技术人员在使用该术语的上下文中不清楚该术语的用法，“大概”和“大约”将表示该特定术语的正负10％，而“实质上”和“显著”将表示该特定术语的正负10％以上。
在本发明上下文中，术语“包括”和“包含”应被解释为开放性的，允许在列举的那些部分的基础上进一步包含附加的部分。术语“组成”和“构成”应解释为封闭性的，不允许包含除权利要求书中列举的部分以外的其他部分。术语“基本上由…组成”应被解释为部分封闭，即只允许包含不会从根本上改变所主张的目标物质性质的附加成分。
情态动词“可以”或“可能”是指在所述的几个实施例或特征中，一个或多个选项或选择的优选使用或选择。如果没有披露关于其中包含的特定实施例或特征的选项或选择，情态动词“可以”指关于如何进行或使用所述实施例或特征的肯定行为。在后面的语境中，情态动词“可以”与助动词“可以”具有相同的含义和内涵。
在本发明上下文中，使用如下的氨基酸缩写：
丙氨酸：Ala(A)       精氨酸：Arg(R)
天冬酰胺：Asn(N)     天冬氨酸：Asp(D)
半胱氨酰：Cys(C)     谷氨酰胺：Gln(Q)
谷氨酸：Glu(E)       甘氨酸：Gly(G)
组氨酸：His(H)       异亮氨酸：Ile(I)
亮氨酸：Leu(L)       赖氨酸：Lys(K)
甲硫氨酸：Met(M)     苯丙氨酸：Phe(F)
脯氨酸：Pro(P)       丝氨酸：Ser(S)
苏氨酸：Thr(T)       色氨酸：Trp(W)
酪氨酸：Tyr(Y)       缬氨酰：Val(V)
在本发明上下文中，术语“肽”、“多肽”和“蛋白质”指包含由酰胺键连接的氨基酸残基的链a聚合物的分子。术语“氨基酸残基”包括但不限于由丙氨酸(Ala或A)、半胱氨酸(Cys或C)、天冬氨酸(Asp或D)、谷氨酸(Glu或E)、苯丙氨酸(Phe或F)、甘氨酸(Gly或G)、组氨酸(His或H)、异亮氨酸(lie或I)、赖氨酸(Lys或K)、亮氨酸(Leu或L)、蛋氨酸(Met或M)、天冬酰胺(Asn或N)组成的组中所含的氨基酸残基，脯氨酸(Pro或P)、谷氨酰胺(Gin或Q)、精氨酸(Arg或R)、丝氨酸(Ser或S)、苏氨酸(Thr或T)、缬氨酸(Val或V)、色氨酸(Trp或W)和酪氨酸(Tyr或Y)残基。术语“氨基酸残基”也可包括非标准或非天然 氨基酸。术语“氨基酸残基”可包括α、β、γ和δ氨基酸。
在本发明上下文中，“蛋白”、“多肽”和“肽”在本文可互换使用，以表示与长度或翻译后修饰(例如，糖基化或磷酸化)无关的、通过酰胺键共价连接的至少两个氨基酸聚合物。
在本发明上下文中使用的“类似物”或“衍生物”是与给定化合物例如肽具有大于约70％但小于100％的序列相似性的化合物，例如肽。这些类似物或衍生物可以包含非天然存在的氨基酸残基以及天然存在的氨基酸残基。这些类似物或衍生物也可以包含一种或多种D-氨基酸残基，并且可以在两个或多个氨基酸残基之间含有非肽连接。
在本发明上下文中，“多核苷酸”是指由核苷酸单位组成的聚合物。多核苷酸包括天然存在的核酸，如脱氧核糖核酸(DNA)和核糖核酸(RNA)，以及核酸类似物。核酸类似物包括非天然存在的碱基、参与除天然存在的磷酸二酯键之外的与其它核苷酸的键的核苷酸、或者包括通过除磷酸二酯键之外的键连接的碱基。因此，核苷酸类似物包括，例如但不限于，硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基磷酸酯、硼烷磷酸酯、膦酸甲酯、手性膦酸甲酯、2-O-甲基核糖核苷酸、肽-核酸(PNA)等。例如可以使用自动DNA合成仪合成这些多核苷酸。术语“核酸”一般是指大的多核苷酸。术语“寡核苷酸”一般是指短的多核苷酸，通常不大于大约50个核苷酸。应当理解，当核苷酸序列用DNA序列(即，A、T、G、C)表示时，这也包括RNA序列(即，A、U、G、C)，其中“U”代替“T”。
根据本发明的一个实施方案，提供含有用于编码根据本发明所述的一种或多种多肽和/或蛋白质的核酸的载体，例如表达载体。
在本发明上下文中，术语“编码”是指诸如基因、cDNA或mRNA等多核苷酸中核苷酸的特定序列作为模板用于合成生物过程中的其它聚合物和大分子的的固有性质，这些聚合物和大分子具有确定的核苷酸序列(即，rRNA、tRNA和mRNA)或确定的氨基酸序列和由其产生的生物学性质。因此，如果在细胞或其它生物系统中，一个基因产生的mRNA的转录和翻译产生一种蛋白质，则该基因编码该蛋白质。用作基因或cDNA的转录模板的编码链(其核苷酸序列与mRNA序列相同并且通常在序列表中提供)和非编码链都可以被称为编码该基因或cDNA的蛋白质或其它产物。除非另外说明，“编码氨基酸序列的核苷酸序列”包括彼此为简并形式并且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列只可以包含内含子。
在本发明上下文中，术语“载体”指能够运输与其连接的另一核酸的核酸分子，“表达载体”是指包含重组多核苷酸的载体，该重组多核苷酸包含与将要表达的核苷酸序列可操 作连接的表达控制序列。表达载体包含足以用于表达的顺式作用元件；其它表达元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域已知的所有表达载体，如粘粒、质粒(例如，裸露的或包含于脂质体中的)和整合了重组多核苷酸的病毒。
在本发明上下文中，术语“重组多核苷酸”是指具有自然界并不连接在一起的序列的多核苷酸。扩增的或装配的重组多核苷酸可以包含在合适的载体中，并且该载体可以用来转化合适的宿主细胞。包含重组多核苷酸的宿主细胞被称为“重组宿主细胞”。然后该基因在重组宿主细胞中表达，以产生例如“重组多肽”。重组多核苷酸也可以发挥非编码功能(例如，启动子、复制起点、核糖体结合位点等)。
在根据本发明的一个实施方案中，表达载体是“质粒”，它指的是一个环形双链DNA环，额外的DNA片段可以连接到该环中。
在本发明上下文中，“插入”是指将一个或多个核苷酸添加到天然多核苷酸序列中。本文公开的工程菌株可包括插入一个或多个基因。在一些实施例中，本文公开的工程菌株包括插入编码外源苯丙氨酸解氨酶(PAL)的序列。在一些实施例中，本文公开的工程菌株包括内源基因中的插入(即基因组插入)，其导致非功能性基因产物。
在本发明上下文中，术语“替换”指将天然多核苷酸序列的核苷酸替换为非该多核苷酸序列天然的核苷酸。本文公开的工程菌株可包括一个或多个基因中的替换。在一些实施例中，替换导致非功能性基因产物，例如，其中替换在基因产物的编码序列中引入过早终止密码子(例如，TAA、TAG或TGA)。在根据本发明的一些实施方案中，根据本发明的工程菌株可包括两个或多个替换，其中替换引入多个过早终止密码子(例如TAATAA、TAGTAG或TGA)。
在本发明上下文中，术语“引物”是指能够与指定的多核苷酸模板特异性杂交并且提供互补多核苷酸的合成起点的多核苷酸。当多核苷酸引物置于诱导合成的条件下时，即，在核苷酸、互补多核苷酸模板和聚合作用试剂如DNA聚合酶的存在下，这种合成发生。引物一般是单链的，但是也可以是双链的。引物一般是脱氧核糖核酸，但是许多种类的合成和天然存在的引物也可用于许多应用。引物与设计与之杂交的模板互补，以用作启动合成的位点，但是不需要反映模板的确切序列。在这种情况下，引物与模板的特异性杂交取决于杂交条件的严格性。引物可以用例如发色、放射性或荧光部分标记，并且用作可检测部分。
在本发明上下文中，术语“野生型”(wild type)是指生物体的自然遗传形式。野生型不同于突变形式(具有遗传突变的生物体)。
在本发明上下文中，“根据本发明的变体”、“PAL变体”、“苯丙氨酸解氨酶的变体”、“变体PAL”、“PAL变体酶”和“PAL酶变体”可互为替换地使用。
在本发明上下文中，术语“有效量”是指足以对个体的健康状况、病理学和疾病产生希望的结果或用于诊断目的的剂量。希望的结果可以包括剂量接受者的主观的或客观的改善。“治疗有效量”是指有效产生对健康的预期有益效果的药物的量。在任意的个别情况中，适当的“有效”量可以由本领域技术人员使用常规实验确定。
在本发明上下文中，术语“治疗”是指预防性治疗或治疗性治疗或诊断性治疗。
在本发明上下文中，术语“药物组合物”是指适合在包括人和哺乳动物的动物个体中作为药物使用的组合物。药物组合物包含药理学有效量的PAL多肽，也包含药学上可接受的载体。药物组合物包括包含活性成分和构成载体的惰性成分，以及由任意两种或多种成分的组合、复合或聚集、或一种或多种成分的解离、或一种或多种成分的其它类型的反应或相互作用直接或间接产生的任何产物。因此，本发明的药物组合物包括通过将本发明的化合物或偶联物与药学上可接受的载体混合制成的任何组合物。
在本发明上下文中，术语“药学上可接受的载体”是指标准药物载体、缓冲液和赋形剂(例如磷酸盐缓冲液、5％葡萄糖水溶液)和乳剂(如油包水型或水包油型乳剂)和各种类型的湿润剂和/或佐剂中的任何一种。
在本发明的第一方面，提供根据本发明的PAL变体、PAL变体酶、PAL突变体或类似物。根据本发明的一个实施方案，所述PAL来源于变异鱼腥藻并具有SEQ:ID NO:1所示的氨基酸序列，且根据本发明的PAL变体具有选自以下的任意一个或两个以上的组合的氨基酸替换：C503S/C565L，C503S/C565P，C503L/C565P，T102R，T102H，I165L，I165V，G166A，G166S，G218A，G218S，G218N，M222L，M222I，N437S，S438N，S438A，I439V，I439C，I439A，I439K，A440T，A440G，A440V，A440T，R441N，R442Q，T445V，T445S，A447S，E448K，N453G，Q457L和G458K。优选地，根据本发明的PAL变体酶具有苯丙氨酸解氨酶活性(EC 4.3.1.24)。优选地，根据本发明的PAL变体酶具有相对于野生型PAL酶增强的苯丙氨酸解氨酶活性(EC 4.3.1.24)。借助于根据本发明的PAL变体，实现了从苯丙氨酸到反式肉桂酸的催化活性的增强、热稳定性的增强和低聚集性。
根据本发明的一个实施方案，根据本发明的PAL变体包含氨基酸取代C503L/C565P/222L，或氨基酸取代C503L/C565P/G218A。
根据本发明的一个实施方案，在苯丙氨酸转化为反式肉桂酸的反应中，所述变体的活性高于包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶。
根据本发明的一个实施方案，述变体与包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶相比，在37℃至70℃范围内的温度下表现出相同的稳定性或更高的稳定性，换句话说，在37℃至70℃范围内的温度下，根据本发明的PAL变体具有比野生型PAL相同或更高，且优选更高的稳定性。
根据本发明的一个实施方案，所述变体与包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶相比具有低聚集性。
根据本发明的一个实施方案，根据本发明的PAL变体或衍生物可具有这样的氨基酸序列，其包括相对于参考氨基酸序列SEQ:ID NO:1的保守氨基酸替换或非保守氨基酸替换。例如，根据本发明的PAL变体或衍生物肽、多肽或蛋白质可包括相对于参考肽、多肽或蛋白质的保守氨基酸替换和/或非保守氨基酸替换。“保守氨基酸替换”是指预计对参考肽、多肽或蛋白质的性质干扰最小的替换，举例来说以下六组中每组包含彼此互为保守性取代的氨基酸：(1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；(2)天冬氨酸(D)、谷氨酸(E)；(3)天冬酰胺(N)、谷氨酸(Q)；(4)精氨酸(R)、赖氨酸(K)；(5)异亮氨酸(I)、亮氨酸(L)、蛋氨酸(M)、缬氨酸(V)；以及(6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。“非保守氨基酸替换”是指预计对参考肽、多肽或蛋白质的性质干扰最大的替换。换句话说，保守氨基酸替换基本上保留了参考肽、多肽或蛋白质的结构和功能，而非保守氨基酸替换不保留参考肽、多肽或蛋白质的结构和功能。
根据本发明的一个实施方案，根据本发明的PAL变体可表达在植物、真菌和细菌中。
根据本发明的一个优选实施方案，根据本发明的PAL变体可表达在点状念珠藻、变异鱼腥藻、圆红冬孢酵母、海洋链霉菌、巢状倒囊藻、发光光杆状菌、轮丝链霉菌、大肠杆菌。
根据本发明的一个实施方案，根据本发明的PAL变体可表达在益生菌或工程菌中，所述益生菌或工程菌能够进行多核苷酸重组并且不是致病菌，也不产生毒素。通过将用于编码根据本发明的PAL变体的重组多核苷酸导入所述益生菌或工程菌体内并使其表达，可以产生根据本发明的PAL变体。所述益生菌或工程菌的实例包括大肠杆菌和G+细菌，例如枯草杆菌。
在本发明的第二方面，本发明提供一种包含根据本发明的PAL变体的缀合物，其中该缀合物包括根据本发明的PAL变体和适合的聚合物，并尤其由根据本发明的PAL变体和适合的水溶性聚合物组成。在此，所述适合的水溶性聚合物包括聚乙二醇、聚两性离子聚合物、聚唑啉。根据本发明的一个优选的实施方案，该水溶性聚合物是聚乙二醇。
在本发明的第三方面，本发明提供一种药物组合物，所述药物组合物包括：(i)根据本发明的PAL变体酶；和(ii)合适的药学上可接受的载体。根据本发明的一个实施方案，根据本发明的药物组合物包括：(i)根据本发明的PAL变体酶的缀合物；以及(ii)合适的药学上可接受的载体。其中可以考虑的是，所述PAL变体酶的缀合物包含与聚乙二醇(PEG)聚合物化学偶联的PAL变体酶。
根据本发明的另一个实施方案，根据本发明的药物组合物中的PAL变体与SEQ ID NO:4具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性。
根据本发明的一个实施方案，配制根据本发明的药物组合物并向需要其的受试者施用。考虑到诸如特定患者的年龄、性别、体重和状况以及给药途径等因素，根据本发明的药物组合物可以按照医学领域的技术人员所熟知的剂量和技术来配制和/或给药。
根据本发明的一个实施方案，根据本发明的药物组合物包含药学上可接受的载体、稀释剂、赋形剂和表面活性剂。此外，所述药物组合物还可包括防腐剂(例如，抗菌剂或抗菌剂，例如苯扎氯铵)。根据本发明的一个优选实施方案，所述药物组合物还可包括缓冲剂(例如，为了将组合物的pH保持在6.5和7.5之间)。
根据本发明的一个实施方案，根据本发明的药物组合物可以用于治疗苯丙酮尿症。
在本发明的第四方面，本发明提供用于编码根据本发明的PAL变体的多核苷酸，该多核苷酸是DNA或RNA，尤其该多核苷酸是cDNA或mRNA或tRNA。这些DNA和RNA具有特定的核苷酸序列，使得以它们为模板可以合成根据本发明的PAL变体酶。
根据本发明的一个实施方案，根据本发明的多核苷酸被密码子优化，使得所述多核苷酸可用于在大肠杆菌中表达根据本发明的PAL变体。根据本发明的一个优选实施方案，根据本发明的多核苷酸被密码子优化，使得所述多核苷酸可用于在动物细胞，尤其人细胞中表达根据本发明的PAL变体。
根据本发明的一个优选实施方案，根据本发明的多核苷酸被密码子优化，使得能够在大肠杆菌中表达根据本发明的PAL变体酶。根据本发明的一个实施方案，根据本发明的多核苷酸被密码子优化，使得能够在人细胞中表达根据本发明的PAL变体酶。公开的多核苷酸可存在于本领域已知的载体(例如质粒载体)中。根据本发明的一个实施方案，PAL酶变体的编码基因在插入载体pET28a时，在酶切位点NcoI自带的起始密码子后引入碱基“GC”，即第一个甲硫氨酸后引入一个甘氨酸，防止翻译时移码。
所述PAL变体例如变异鱼腥藻苯丙氨酸解氨酶PAL(SEQ ID NO:1)的变体，例如包括 SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列的PAL变体的其他根据本发明的PAL变体。
在本发明的第五方面，提供一种根据本发明的表达载体，该表达载体包含根据本发明的多核苷酸，其中，所述表达载体可以在活细胞中表达。
根据本发明的一个实施方案，根据本发明的表达载体包括本领域已知的所有表达载体，如粘粒、质粒(例如，裸露的或包含于脂质体中的)和整合了重组多核苷酸的病毒。
根据本发明的一个优选实施方案，根据本发明的表达载体是病毒载体，例如腺病毒，腺相关病毒以及慢病毒。
根据本发明的另一个优选实施方案，根据本发明的表达载体是质粒，该质粒包含启动子，并且该启动子与根据本发明的多核苷酸可操作地连接。根据本发明的又一个优选实施方案，所述质粒通过脂质体或阳离子聚合物转染进入细胞。
在本发明的第六方面，本发明提供一种根据本发明的细菌，其中所述细菌在基因组上整合有根据本发明的多核苷酸和/或能够表达根据本发明的PAL变体。在此，本发明还提供一种根据本发明的修饰细胞，所述修饰细胞包括根据本发明的PAL变体和/或根据本发明的多核苷酸和/或根据本发明的表达载体，其中任选的，该修饰细胞是动物细胞，尤其是人细胞。
在包含用于编码至少一种根据本发明的PAL变体的至少一种多核苷酸的宿主细胞(例如根据本发明的细菌或根据本发明的修饰细胞)中，所述多核苷酸可操作地连接到用于在宿主细胞中表达PAL变体的一个或多个控制序列。适合于用于表达根据本发明的PAL变体的宿主细胞例如根据本发明所述的细菌，诸如大肠杆菌、河流弧菌(Vibriofluvialis)、链霉菌属和鼠伤寒沙门氏菌(Salmonellatyphimurium)细胞；真菌细胞，诸如酵母细胞(例如，酿酒酵母或毕赤酵母(Pichiapastoris)；昆虫细胞，诸如果蝇属(Drosophila)S2和夜蛾属(Spodoptera)Sf9细胞；动物细胞，诸如CHO、COS、BHK、293和Bowes黑素瘤细胞；和植物细胞。示例性的宿主细胞还包括各种大肠杆菌(Escherichiacoli)菌株(例如，W3110(ΔfhuA)和BL21(DE3))
在本发明的第七方面，本发明提供一种制备根据本发明的PAL变体的方法，所述方法包括在培养基中培养根据本发明的细菌或根据本发明的修饰细胞以产生根据本发明的PAL变体并从所述修饰细胞和/或所述培养基中分离根据本发明的PAL变体。
通过根据本发明的方法获得的PAL变体酶可用于治疗以苯丙氨酸血浓度升高为特征的疾病或紊乱，例如苯丙酮尿症(PKU)。
根据本发明的一个实施方案，通过根据本发明的方法获得的PAL变体酶相比于来源于变异鱼腥藻的PAL酶(SEQ ID NO:1)具有选自以下的一项或多项的氨基酸替代：C503S/C565L，C503S/C565P，C503L/C565P突变以及T102R，T102H，I165L，I165V，G166A，G166S，G218A，G218S，G218N，M222L，M222I，N437S，S438N，S438A，I439V，I439C，I439A，I439K，A440T，A440G，A440V，A440T，R441N，R442Q，T445V，T445S，A447S，E448K，N453G，Q457L和G458K。
具体地，根据本发明的一个实施方案，根据本发明的方法具体包括以下一个或多个步骤：(i)构建包括质粒、表达细胞或类似物的PAL原始酶表达体系；(ii)从PAL宿主细胞中提取pET28a-PAL质粒；(iii)使用从步骤(ii)中获得的质粒作为模板，利用定点突变技术构建突变库；(iv)使用高通量筛选方法培育PAL变体，并通过检测变体酶活的方式筛选优突变。
本发明上下文中的组分可以是粗品和/或至少部分地经过分离和/或纯化。在本发明上下文中，术语“分离或纯化”可指从其自然环境和/或介质中取出的组分，且至少60％游离，优选至少75％游离，且更优选至少90％游离，甚至更优选至少95％游离于它们与之天然相关联的其他组分和/或介质。
在根据本发明的用于制备根据本发明的PAL变体的方法中，适用于所述方法的PAL酶变体包括但不限于来源于变异鱼腥藻的PAL酶的变体。合适的转化细胞可包括但不限于转化的大肠杆菌，其中PAL基因序列优选地已被密码子优化以在大肠杆菌中表达根据本发明的PAL酶变体。
根据本发明的一个实施方案，可通过进一步补充和/或修改培养基以促进转化细胞生长。根据本发明的一个实施方案，在培养基中添加葡萄糖，可选浓度为0.1-0.3％(v/v)。
根据本发明的一个实施方案，转化细胞可以传代一次或多次。例如，可在培养基达到至少约1-2的OD600(例如，当转化细胞仍处于生长阶段时)后，将转化细胞从培养基中移出，并将转化细胞置于添加有苯丙氨酸的新鲜的最小基本培养基(Minimum Essential Medium)中。这个可选的继代培养步骤可以执行一次或多次。任选地，当培养物包含过量tCA时，例如，当培养基包含至少0.5、1.0、1.5或2.0mM tCA时，执行传代培养步骤以从培养物中移出转化细胞。
在本发明的第八方面，本发明提供一种在有需要的受试者中治疗疾病或病症的方法，所述疾病或病症的特征在于苯丙氨酸的水平升高，其中，所述方法包括向受试者施用根据本发明第一方面所述的PAL变体；根据本发明第二方面所述的缀合物；根据本发明第三方 面所述的药物组合物；根据本发明第四方面所述的多核苷酸；根据本发明第五方面所述的表达载体和/或根据本发明第六方面所述的细菌和修饰细胞。
根据本发明的一个实施方案，在根据本发明在有需要的受试者中治疗疾病或病症的方法中，以本领域已知的任何合适的方法进行给药，例如口腔、经皮、透粘膜、肺内(包含雾化)、肌内、皮下或静脉内给药。根据本发明的一个优选实施方案，通过皮下或者口服给药。
根据本发明的一个实施方案，以本领域已知的方式选择剂型用于口服或肠胃外制剂。根据本发明的药物组合物可以是例如，固体、半固体或者液体剂型的形式，如片剂、栓剂、丸剂、胶囊、粉末、液体、混悬液、乳膏、软膏、洗液或类似物，例如适于精确剂量单一给药的单位剂型。根据本发明的药物组合物可以包括结合有药学上可接受的载体的治疗有效量的根据本发明的PAL变体；此外，可以可选地包括其他药剂、佐剂、稀释液、缓冲液等等。
根据本发明的一个实施方案，可以使用缓释或者持续释放系统，以便保持恒定的剂量水平。
根据本发明的一个实施方案，对所述受试者皮下施用根据本发明第一方面所述的PAL变体或根据本发明第二方面所述的缀合物，以治疗以苯丙氨酸的水平升高为特征的疾病和病症，特别地，所述疾病或病症是苯丙酮尿症(PKU)。
根据本发明的一个实施方案，对所述受试者皮下施用根据本发明第二方面所述的缀合物，从而在向有需要的受试者施用时增加PAL变体酶的半衰期。根据本发明的一个实施方案，所述缀合物包含与聚乙二醇(PEG)聚合物化学偶联的PAL变体酶。
附图简述
为使本发明的上述目的、特征和优点能够更加容易理解，下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，因此本发明不受下面公开的具体实施例的限制。
图1示出了苯丙氨酸解氨酶(PAL)或PAL变体催化苯丙氨酸生成反式肉桂酸示意图；
图2A示意性示出了PAL四聚体中单个亚基的三维结构；
图2B示意性示出了部分突变位点在三维结构上位置；
图3A和图3B示意性示出了根据本发明PAL高通量筛选位点与具备苯丙氨酸解氨酶活性的氨基酸替代；
图4示意性示出了PAL与其变体(PAL201:C503L/C565P；PAL202:G218A/C503L/C565P；PAL204：M222L/C503L/C565P)经组合纯化后SEC-HPLC图谱(SEC层析柱Ultrahydrogel 1000(Waters)，流动相：0.3ml/min PBS缓冲溶液)；
图5示意性示出了根据本发明的部分PAL变体经亲和层析纯化后的SDS-PAGE图；
图6示意性示出了根据本发明的PAL变体C503L/C565P在pH值为6.5-9.0之间的酶活变化；
图7示意性示出了根据本发明的PAL变体与野生型PAL的酶活在温度为37-70℃的热稳定性变化；
图8示意性示出了根据本发明的部分PAL变体在经聚乙二醇(PEG)修饰前后的HPLC-SEC图谱。
具体实施方式
如附图所中所阐释和举例的，从以下对具体实施方案的更详细描述，上述和其他目的、元件和优点将变得明显。就其所使用范围而言，如在本文的权利要求书和说明书中所使用的术语“一个”、“一种”和单数形式的词应理解为包括相同词的复数形式，使得这些术语表示提供了一个或多个某物。术语“至少一个”和“一个或多个”可以互换使用。术语“单个”应用于指示意指某物的一个且仅一个。类似地，当需要特定数量的事物时，将使用其他特定的整数值，例如“二”。术语“优选地”、“优选的”、“优选”、“任选地”、“可以”以及类似的术语用于指示所引述的项目、条件或步骤是实施方案中的可选的(即，不是必需的)的特征。除非另有说明，否则描述为“在a与b之间”的范围包括“a”和“b”的值。
实施例1基因工程菌E.coli BL21(DE3)/pET28a-PAL的构建
将源自变异鱼腥藻的苯丙氨酸解氨酶PAL编码基因(氨基酸序列如SEQ ID NO.1所示)进行密码子优化，密码子优化后的PAL基因的核苷酸序列如SEQ ID NO:2所示。密码子优化后的PAL基因的核苷酸序列(SEQ ID NO.2)经人工合成后插入pET28a的Nco I和Xho I之间得到重组质粒pET28a-PAL，电泳图见图2所示。
取出5μL质粒pET28a-PAL，加入到含50ul E.coli BL21(DE3)感受态细胞的1.5ml EP管中，轻弹管壁混匀，冰上放置30min。42℃水浴热击45s后，立即置于冰上2min。向EP管中加入1mL LB液体培养基，在37℃，培养1h。将培养液4500rpm离心4min。取出800μL上层清液，用剩余上层清液重悬菌体，取出100μL涂布在含有50μg/mL卡那霉素的LB固体培养基上。在37℃下培养12-14h，得到重组表达PAL的工程菌E.coli BL21(DE3)/pET28a-PAL。
LB液体培养基组成：酵母提取物5g/L，胰蛋白胨10g/L，NaCl 10g/L，溶剂为蒸馏水，pH为约7.0。
实施例2 pET28a-PAL饱和突变质粒和含有突变质粒的表达菌构建
以实施例1制备的质粒pET28a-PAL为模板，设计一对引物，并将突变位点引物序列设置为“NNN”，利用反向PCR技术扩增全质粒，加入DpnI消化质粒，利用试剂盒(TaKaRa)纯化质粒，利用电转化方法将质粒导入克隆宿主菌DH5α中，质粒经过克隆菌富集后，提取质粒，再将质粒转化到大肠杆菌E.coli BL21(DE3)。各个点饱和突变的引物见表1。
表1点饱和突变引物

反向PCR扩增体系见表2所示。
表2 PCR扩增体系
PCR反应进程如下，PCR产物经0.8％琼脂糖凝胶电泳检测。
表3 PCR扩增条件
将得到的饱和突变质粒取出5μL加入到50μL E.coli Top10感受态中，轻弹管壁混匀，冰上放置30min。42℃水浴热击45s，立即置于冰上2min。向管中加入1mL LB液体培养基，37℃，培养1h。将培养液4500rpm离心4min，取出800μL上层清液。用剩下的上层清液液重悬菌体，取出100μL涂在含有100μg/mL卡那霉素的LB固体培养基上。37℃， 培养12-16h，得到含有突变质粒的克隆菌，克隆菌富集培养后提取质粒，将质粒转化到E.coli BL21(DE3)感受态细胞中，重复上述转化步骤，即得到含有突变质粒的表达菌。
实施例3：PAL突变质粒表达菌的高通量筛选
1、菌体培养
从平板上挑取突变质粒表达菌(含有或不含有His标签)，接种到96深孔板中，培养基为LB/Kana+，在37℃下以800rpm培养16h，取出100ul转接到另一块96深孔板，每孔有900ulTB培养基，已经添加了Kana和IPTG，在37℃下以800rpm培养16h，然后在4℃下以3000rpm离心30min，移除上层清液，获得湿菌体细胞。
2、菌体细胞破碎
将96深孔板中的湿菌体细胞在温度为约-80℃的冰箱中冻融两次，然后用含有溶菌酶的Tris-HCl缓冲液(pH＝8.5)重悬菌体细胞，在37℃下，以800rpm震荡30min，相同条件下离心，上层清液液即为粗酶液。
3、酶活检测与酶活计算
取10ul粗酶液，加入190ul含有22.5mM苯丙氨酸的Tris-HCl(100mM，pH8.5)缓冲液进行测定，使用其中空载质粒孔和PAL孔作为对照组，剩余孔作为实验组，测定波长是290nm，参数设定为测吸光度，共测定10min，每隔30s测定一次数据。
酶活单位定义：在37℃以及pH 8.5条件下，每分钟内将1u mole L-苯丙氨酸脱氨基转化为反式肉桂酸与NH3定义为1U。
酶活计算公式如下：
酶活(U/ml)＝Δc×V1/(t×V2)
Δc：产物浓度变化，单位uM，通过建立产物浓度与吸光度之间的线性关系，代入得到检测的产物浓度变化；
V1：测活体系的总体积，单位是ml；
t：测活总时长，单位是min；
V2：加入酶液的体积，单位是ml
4、PAL变体酶与野生型PAL酶的酶活对比
对比结果见表4，其中“+”表示PAL变体的酶活高于野生型PAL酶，“o”表示酶活持平于野生型PAL酶，“-”表示酶活低于野生型PAL酶。
表4 PAL变体酶与野生型PAL酶的酶活对比
实施例4：pET28a-PAL及其突变质粒表达菌菌体培养与蛋白诱导
pET28a-PAL及其突变质粒转化到E.coli BL21中后，得到野生型PAL与PAL变体表 达菌，从平板上挑取单菌落到LB/Kana液体培养基，在37℃下，以200rpm过夜培养12-16h，将菌液转移到新的LB/Kana液体培养基中，接种比例为2％，在37℃下以200rpm培养1-2h，等到OD600在0.6-0.8之间时，加入终浓度为0.2mM的IPTG，在30℃下以200rpm诱导12-16h。
诱导结束后，采用离心法收菌，在4℃下以6000rpm离心10min。湿菌体在-80℃下保存。
实施例5：蛋白比活测定
蛋白浓度检测：蛋白测量的方法引用BCA检测试剂盒的(Thermo)方法，该方法以引用的方式并入本文；其中线性的范围在25ug/ml至2000ug/ml。本实施案例采用96孔检测板，添加的酶液和检测液体积分别是10ul和200ul，或者25ul和200ul。
酶活性检测：如实施例3所述酶活检测方法，检测野生型PAL和PAL变体的酶活。
比活计算：
比活(U/mg)＝酶活(U/ml)/蛋白浓度(mg/ml)
实施例6：野生型PAL和PAL变体的纯化
细胞裂解：将冷冻过的细胞团融化并再悬浮于室温下pH值为8.0的20mM Tris-HCl、100mM NaCl的缓冲液中，以形成密度为约120至140OD600的细胞浆料。将细胞两次通过700-800Bar的Niro NS30006均质机(其中温度控制在30℃以下)以通过均化作用裂解细胞。均化作用之后，通过添加1N NaOH至裂解液，将pH值调整为约8.0。将细胞裂解物渐渐加热到55℃，保持55℃30至120min，然后冷却。离心澄清细胞裂解液。将包含目标酶的裂解液上层清液液加入硫酸铵至饱和度30-70％，静置过夜。离心收集沉淀。以pH 8.5的Tris缓冲溶液重悬酶沉淀。
蛋白纯化：
1.包含His标签的PAL及其变体：
用5mM咪唑(50mMTris-HCl，pH8.5，0.5MNaCl平衡Ni-NTA亲和层析柱2-5个柱体积；将经0.45um滤膜过滤的细胞破碎液或上述Tris重悬液载入层析柱；以咪唑浓度为5mM-200mM(50mMTris-HCl，pH8.5，0.5MNaCl)的缓冲溶液梯度洗脱，收集洗脱峰，用SDS-PAGE检测融合蛋白的分子量大小和纯度。部分PAL变体的结果在图5中示出，其中，图5示出共9个泳道，其中从左到右依次为蛋白标样Marker(第1泳道)和包含His标签的PAL变体(第2至第9泳道)，其中第2至第9泳道依次代表：包含C503L/C565P突变的PAL变体、包含突变T102R/C503L/C565P的PAL变体、包含G218A/C503L/C565P突变的PAL变体、 包含G218S/C503L/C565P突变的PAL变体、包含M222L/C503L/C565P突变的PAL变体、包含S438N/C503L/C565P突变的PAL变体、包含I439V/C503L/C565P突变的PAL变体；其中示出的标样条带从上到下依次为200、140、95、65、52、41、33、25、17、10。可以看出，目标PAL变体在相对分子量52-65kDa之间出现；
2.不包含His标签的PAL及其变体：
将Tris重悬酶液依次穿过疏水作用(HIC)柱和阴离子交换(AIEX)柱，以纯化得到所述PAL变体。应当注意的是，疏水作用(HIC)柱和阴离子交换(AIEX)柱的使用顺序可以进行交换，也可以只使用一种色谱柱作为纯化工具。应当理解的是，可以使用其他AIEX和HIC柱树脂，以及所述HIC柱可以替换为体积排阻色谱法。用pH值为8.5的50mM Tris-HCl缓冲溶液稀释沉淀重悬酶液至2倍，并装入经pH值为8.5的50mM Tris-HCl缓冲溶液平衡的TPGQ AIEX柱。以pH值为8.5的50mM Tris-HCl、400mM NaCl缓冲溶液冲洗柱，并利用含有0至1000mM NaCl的Tris-HCl缓冲液(50mM，pH 8.5)梯度洗脱PAL。收集具有酶活性的组分，并超滤和浓缩。疏水柱层析：将HIC层析柱(t-Butyl，东曹)用含有1M(NH4)2SO4的Tris-HCl缓冲溶液(50mM，pH 8.5)预先平衡。将PAL酶液用含有2M(NH4)2SO4的Tris-HCl缓冲溶液(50mM，pH 8.5)稀释2倍，并装入上述HIC层析柱，并以含有1M至0M(NH4)2SO4的Tris-HCl缓冲溶液(50mM，pH 8.5)梯度洗脱，收集具有酶活的组分，并进行超滤浓缩脱盐。
野生型PAL酶与部分PAL变体酶经纯化后，得到的比活数据总结在表5中。
表5.野生型PAL酶与PAL变体酶的比活
实施例7：野生型PAL酶与PAL变体酶的聚集性比较
浓缩纯化的PAL，并将浓缩的蛋白质溶液(2.5mg/ml)在37℃下孵育2小时，加速纯化的PAL蛋白质在溶液中的聚集。通过SEC-HPLC分离PAL蛋白来检测聚集性。为了确定 二硫键交联是否为聚集的原因，将50mM二硫苏糖醇(DTT)添加到浓缩蛋白质溶液中，然后在37℃下孵育2小时。
对于易于聚集的野生型PAL溶液通过超滤进一步浓缩至约10mg/mL，并在37℃下孵育2小时。对于抗聚集的PAL变体，将溶液进一步浓缩至约30mg/mL，并在37℃下孵育2小时。
如表6所示，纯化的野生型PAL酶在37℃下孵育2小时后出现聚集。正如预期的那样，当PAL蛋白在37℃下孵育2小时之前浓缩时，这种聚集会加剧。浓缩的蛋白质暴露于DTT中，可阻止聚集，表明聚集是由二硫键交联引起的。相比之下，纯化的PAL变体(C503S/C565P)和PAL变体(C503S/C565L)等在37℃下培养2小时后没有形成聚集体，这表明PAL变体与野生型PAL酶相比具有更低的聚集性。
表6.PAL变体的不同处理对于形成聚集物的影响

其中“-”表示没有出现聚集，“+”表示出现聚集，“++”表示出现剧烈的聚集。
实施例8：野生型PAL酶与PAL变体酶的pH适应曲线
野生型PAL酶和所选PAL变体的最佳pH值通过实施例5所述比活测定来确定。取10ul纯酶液(总蛋白0.2μg)+190ul溶解22.5mM Phe的100mM Tris-HCl(pH8.5)缓冲液进行测定，在37℃下，在200μL体系中进行酶活检测。
其中溶解22.5mM Phe的100mM Tris-HCl(pH8.5)缓冲液分别设置pH值为6.5、7.0、7.5、8.0、8.5、9.0。
测量结果总结在图6中，其中所有结果以具有C503L/C565P突变的PAL变体在pH为7.5时的酶活进行归一化处理。从图6中可以看出，具有C503L/C565P突变的PAL变体在7.5至8.5的pH范围内具有最佳的酶活。
实施例9：野生型PAL酶与PAL变体酶的温度稳定性
温度对野生型PAL酶和所选PAL变体酶的稳定性的影响通过在37℃至70℃的温度范围内将蛋白质在pH7.5的50mM Tris-HCl中培养2小时，然后测量酶活性来确定。每种酶反应均以1μg PAL蛋白和22.5mM苯丙氨酸为底物，在37℃下总反应体积为200μL。
测量结果总结在图7中。所有结果以具有M222L/C503L/C565P突变的PAL变体在37℃时的酶活进行归一化处理。
实施例10：PEG修饰PAL变体
将含有PAL蛋白的溶液在pH＝8.0,10mM的PBS缓冲液透析换液，固定最终蛋白浓度为2.5mg/ml。将分子量为20kDa的PEG(PEG-NHS)加入到上述PAL溶液中，固定最终PEG浓度为75mg/ml。PEG与PAL的反应溶液在室温反应2-4小时，或在4℃下反应过夜。在反应终止时立即取10μl上述反应溶液进行稀释，并依据实施例5中的方法测定酶活，并计算PAL与PEG反应前后的酶活变化(见表7)。将包含PEG-PAL缀合物的反应溶液至于透析袋中(截留分子量300kD)，在PBS 7.4缓冲溶液中充分透析。最终产物通过HPLC-SEC进行检测。如图8所示，三种PAL变体在经PEG修饰后，其在HPLC-SEC图谱中均表现出了明显的左移，表明PAL变体与PEG的成功缀合偶联，整体的分子尺寸显著增加。
表7.PAL变体经PEG修饰后的酶活变化
在根据本发明的实施方案中值得注意的是，所述方法的步骤可以以任何适当的顺序执行，除非本文另有说明或与上下文明显矛盾。除非本文另有说明或上下文另有明确矛盾，否则这些步骤可以重复或重复任意次数以实现预期目标。
使用本文提供的任何及所有示例或示例性语言(例如，“例如”)仅旨在更好地说明本发明，且不构成对本发明范围的限制，除非另有要求。说明书中的任何语言都不应被解释为表明任何非权利要求保护的元素对本发明的实施至关重要。
本文描述了本发明的优选方面，包括发明人已知的用于实施本发明的最佳模式。在阅读前述描述后，这些优选方面的变化对于本领域的普通技术人员来说可能变得显而易见。发明人期望本领域的普通技术人员在适当的情况下采用这种变化，并且发明人希望本发明在本文具体描述的情况以外的情况下实施。
尽管已经参照本发明的特定的实施方案在本文中描述各种改进，应当理解，这种描述仅仅是说明的方式，而不应被解释为限制任何要求保护的发明的范围。因此，任何要求保护的本发明的范围和内容将仅由所附权利要求书的条款以当前形式或如在审查期间修改的形式或在任何继续申请中实行的形式限定。此外，应理解，除非另有说明，否则本文中讨论的任何具体实施方案的特征可以与本文中以其他方式讨论或考虑的任何一个或多个实施方案的一个或多个特征组合。本领域普通技术人员将认识到，在不脱离如所附权利要求中描述的本申请的精神和范围的情况下，可对所描述的实施方式进行一些修改和改变，这些修改和改变落入本发明的保护范围内。

Claims (28)

  1. 一种苯丙氨酸解氨酶(PAL)的变体，该苯丙氨酸解氨酶(PAL)来源于变异鱼腥藻并包含SEQ ID NO:1所示的氨基酸序列，其特征在于，所述变体包含：
    氨基酸取代C503S/C565L，使得所述变体包含SEQ ID NO:2所示的氨基酸序列；或
    氨基酸取代C503S/C565P，使得所述变体包含SEQ ID NO:3所示的氨基酸序列；或
    氨基酸取代C503L/C565P，使得所述变体包含SEQ ID NO:4所示的氨基酸序列。
  2. 根据权利要求1所述的苯丙氨酸解氨酶的变体，其特征在于，所述变体另外包含以下一个或多个氨基酸取代点突变：T102R、T102H、I165L、I165V、G166A、G166S、G218A、G218S、G218N、M222L、M222I、N437S、S438N、S438A、I439V、I439C、I439A、I439K、A440T、A440G、A440V、A440T、R441N、R442Q、T445V、T445S、A447S、E448K、N453G、Q457L和G458K。
  3. 根据权利要求1所述的苯丙氨酸解氨酶的变体，其特征在于，所述变体包含：
    氨基酸取代C503L/C565P/222L，或氨基酸取代C503L/C565P/G218A。
  4. 根据权利要求1或2所述的苯丙氨酸解氨酶的变体，其特征在于，在苯丙氨酸转化为反式肉桂酸的反应中，所述变体的活性高于包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶。
  5. 根据权利要求1或2所述的苯丙氨酸解氨酶的变体，其特征在于，所述变体与包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶相比，在37℃至70℃范围内的温度下表现出相同的稳定性或更高的稳定性。
  6. 根据权利要求1或2所述的苯丙氨酸解氨酶的变体，其特征在于，所述变体与包含SEQ ID NO:1所示的氨基酸序列的苯丙氨酸解氨酶相比具有低聚集性。
  7. 一种缀合物，其特征在于，该缀合物包括根据权利要求1或2所述的苯丙氨酸解氨酶的变体和聚合物，其中该聚合物为水溶性聚合物。
  8. 根据权利要求7所述的缀合物，其特征在于，所述水溶性聚合物为聚乙二醇、聚两性离子聚合物和聚唑啉中的任意一种或两种以上的混合物。
  9. 一种药物组合物，包括：
    (i)根据权利要求1或2所述的苯丙氨酸解氨酶的变体或根据权利要求6所述的缀合物；和
    (ii)合适的药学上可接受的载体，该载体为选自Tris-HCl、NaCl、L-苯丙氨酸(L-Phe)、甘氨酸(Gly)和反式肉桂酸中的任意一种或两种以上的混合物。
  10. 根据权利要求9所述药物组合物，其特征在于，所述药物组合物可以用于治疗苯丙酮尿症。
  11. 用于编码根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体的多核苷酸，其特征在于，所述多核苷酸是DNA或RNA。
  12. 根据权利要求11所述的多核苷酸，其特征在于，其中所述多核苷酸被密码子优化，使得所述多核苷酸可用于在大肠杆菌中表达苯丙氨酸解氨酶的变体。
  13. 根据权利要求11所述的多核苷酸，其特征在于，其中所述多核苷酸被密码子优化，使得所述多核苷酸可用于在动物细胞，尤其人细胞中表达苯丙氨酸解氨酶的变体。
  14. 一种表达载体，其特征在于，该表达载体包含根据权利要求11至13中任一项所述的多核苷酸，其中，所述表达载体可以在活细胞中表达。
  15. 根据权利要求14所述的表达载体，其特征在于，所述表达载体是病毒，所述病毒包括腺病毒，腺相关病毒以及慢病毒。
  16. 根据权利要求14所述的表达载体，其特征在于，所述表达载体是质粒，该质粒包含启动子，并且该启动子与根据权利要求11至13中任一项所述的多核苷酸可操作地连接。
  17. 根据权利要求16所述的表达载体，其特征在于，所述质粒通过脂质体或阳离子聚合物转染进入细胞。
  18. 一种细菌，其特征在于，所述细菌在基因组上整合有根据权利要求11所述的多核苷酸和/或能够表达根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体。
  19. 一种修饰细胞，包括：
    根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体；
    根据权利要求11至13中任一项所述的多核苷酸；
    根据权利要求14至17中任一项所述的表达载体；和
    任选地，所述修饰细胞是修饰的动物细胞。
  20. 根据权利要求19所述的修饰细胞，其特征在于，所述修饰细胞是人细胞。
  21. 一种用于制备苯丙氨酸解氨酶的变体的方法，其特征在于，该方法包括在培养基中培养根据权利要求18所述的细菌或根据权利要求19所述的修饰细胞以产生苯丙氨酸解氨酶的变体并从所述修饰细胞和/或所述培养基中分离所述苯丙氨酸解氨酶的变体。
  22. 根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体在制备用于治疗个体苯丙氨酸水平升高的药物中的用途。
  23. 根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体在制备用于治疗苯丙酮尿症的药物中的用途。
  24. 一种在有需要的受试者中治疗疾病或病症的方法，所述疾病或病症的特征在于苯丙氨酸的水平升高，其特征在于，所述方法包括向所述受试者施用根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体；根据权利要求7或8所述的缀合物；根据权利要求9或10所述的药物组合物或根据权利要求11至13中任一项所述的多核苷酸；根据权利要求14至17中任一项所述的表达载体；根据权利要求18所述的细菌，和/或根据权利要求19或20所述的修饰细胞。
  25. 根据权利要求24所述的方法，其中对所述受试者皮下施用权利要求1至6中任一项所述的变体或权利要求7或8所述的缀合物。
  26. 根据权利要求24所述的方法，其特征在于，所述疾病或病症是苯丙酮尿症。
  27. 根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体，其中所述变体可表达在植物、真菌和细菌中。
  28. 根据权利要求1至6中任一项所述的苯丙氨酸解氨酶的变体，其中所述变体可表达在点状念珠藻、变异鱼腥藻、圆红冬孢酵母、海洋链霉菌、巢状倒囊藻、发光光杆状菌、轮丝链霉菌、大肠杆菌中。
PCT/CN2023/077574 2022-07-18 2023-02-22 Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法 WO2024016658A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210839871.7A CN117417925A (zh) 2022-07-18 2022-07-18 Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法
CN202210839871.7 2022-07-18

Publications (1)

Publication Number Publication Date
WO2024016658A1 true WO2024016658A1 (zh) 2024-01-25

Family

ID=89525274

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/077574 WO2024016658A1 (zh) 2022-07-18 2023-02-22 Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法

Country Status (2)

Country Link
CN (1) CN117417925A (zh)
WO (1) WO2024016658A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101842482A (zh) * 2007-05-25 2010-09-22 生物马林药物股份有限公司 原核生物苯丙氨酸氨裂合酶的组合物及使用所述组合物的方法
CN105324483A (zh) * 2013-04-18 2016-02-10 科德克希思公司 工程化苯丙氨酸解氨酶多肽
CN106497905A (zh) * 2016-12-14 2017-03-15 江南大学 一株鱼腥藻来源的苯丙氨酸脱氨酶的突变体
CN112672989A (zh) * 2018-07-12 2021-04-16 科德克希思公司 工程化苯丙氨酸氨裂合酶多肽
WO2021168060A1 (en) * 2020-02-19 2021-08-26 Trustees Of Tufts College Methods for engineering amino acid ammonia lyase enzymes and enzymes thereby obtained

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101842482A (zh) * 2007-05-25 2010-09-22 生物马林药物股份有限公司 原核生物苯丙氨酸氨裂合酶的组合物及使用所述组合物的方法
CN105324483A (zh) * 2013-04-18 2016-02-10 科德克希思公司 工程化苯丙氨酸解氨酶多肽
CN106497905A (zh) * 2016-12-14 2017-03-15 江南大学 一株鱼腥藻来源的苯丙氨酸脱氨酶的突变体
CN112672989A (zh) * 2018-07-12 2021-04-16 科德克希思公司 工程化苯丙氨酸氨裂合酶多肽
WO2021168060A1 (en) * 2020-02-19 2021-08-26 Trustees Of Tufts College Methods for engineering amino acid ammonia lyase enzymes and enzymes thereby obtained

Also Published As

Publication number Publication date
CN117417925A (zh) 2024-01-19

Similar Documents

Publication Publication Date Title
JP2686090B2 (ja) 新規融合蛋白質およびその精製方法
Batey et al. Interaction of theBacillus stearothermophilusRibosomal protein S15 with 16 S rRNA: I. Defining the minimal RNA site
EP3556854A1 (en) Heat-resistant reverse transcriptase mutant
US20080293102A1 (en) Compositions and methods for producing apolipoprotein
Kato et al. RecA protein from an extremely thermophilic bacterium, Thermus thermophilus HB8
KR20130084323A (ko) Ｏａｓ1 유전자에서의 돌연변이
CN107858340B (zh) 高催化活性的d-果糖-6-磷酸醛缩酶a突变体、重组表达载体、基因工程菌及其应用
TWI465460B (zh) 用於治療病毒感染及癌症之新穎醫藥組合物
US6093396A (en) Modified glutamic acid decarboxylase (GAD)
JP6267285B2 (ja) 治療用組換えグリシンｎ−アシルトランスフェラーゼ
WO2024016658A1 (zh) Pal变体、包含该pal变体的药物组合物以及用于制备该pal变体的方法
CN108977455B (zh) 用于生产草酸脱羧酶的重组质粒、大肠杆菌表达系统及方法和应用
JP2010512168A (ja) 組換えヒトアルギナーゼｉについての改善された発現系
US20230183712A1 (en) Methods for engineering amino acid ammonia lyase enzymes and enzymes thereby obtained
Skehel et al. NADH: ubiquinone oxidoreductase from bovine heart mitochondria Complementary DNA sequence of the import precursor of the 10 kDa subunit of the flavoprotein fragment
KR101778878B1 (ko) 박테로이데스 속 미생물 유래의 ｇａｂａ 생성 고활성 글루탐산탈탄산효소 및 이의 용도
JP2021501603A (ja) β−ラクタマーゼ変異体
JPH1156362A (ja) ロドスポリジウム属由来のｄ−アミノ酸オキシダーゼ
KR101666934B1 (ko) 개량형 TAT 펩타이드가 융합된 EGF, 티모신β4, hGH 단백질의 생산방법 및 이들 단백질을 포함하는 화장료 조성물
RU2740715C2 (ru) Модифицированный белок GroEL микроорганизма Thermus thermophilus
MX2014009979A (es) Metodo para la reduccion del desplazamiento de los marcos de lectura 1->3.
EP4257689A1 (en) Novel promoter variant for constitutive expression and use thereof
WO2018039081A1 (en) Bovine fibroblast growth factor 21 and ketosis in dairy cattle
CN114716563B (zh) 一种融合蛋白及其制备与应用
CN117586997A (zh) 粘红酵母来源的苯丙氨酸解氨酶突变体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23841736

Country of ref document: EP

Kind code of ref document: A1