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• • 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
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
• • 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
  • A61K38/46—Hydrolases (3)
  • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01045—Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase

Definitions

• the present invention relates to a glycosylated protein having glucocerebrosidase activity.
• Lysosomal disease is a hereditary disease caused by decreased activity or deficiency of lysosomal enzymes and their related factors, resulting in the accumulation of substances that act as substrates for these enzymes in the body.
• glucocerebrosidase ⁇ -glucosidase
• GBA glucocerebrosidase
• GBA ⁇ -glucosidase
• Symptoms and findings such as anemia, thrombocytopenia, bone changes, and increased blood acid phosphatase and angiotensin-converting enzyme levels associated with hyperfunction are observed (Non-Patent Document 1).
• enzyme replacement therapy is often used as a treatment method for such lysosomal diseases.
• a recombinant cDNA encoding human glucocerebrosidase expressed in a Chinese hamster ovary (CHO) cell line was glycoengineered to facilitate uptake into target cell macrophages.
• CHO Chinese hamster ovary
• an object of the present invention is to provide a glycosylated protein having glucocerebrosidase activity.
• the present inventors have conducted extensive studies. As a result, the glycosylated protein having glucocerebrosidase activity, which is obtained by adding a sugar chain having a single structure to a protein having glucocerebrosidase activity and having no sugar chain added thereto, was found to We have found that the problem can be solved, and have completed the present invention.
• X to Y includes X and Y and means "X or more and Y or less”. Unless otherwise specified, measurements of operations and physical properties are performed under the conditions of room temperature (20 to 25° C.)/relative humidity of 40 to 50% RH.
• One embodiment of the present invention is a glycosylated protein having glucocerebrosidase activity, which is obtained by adding a sugar chain having a single structure to a protein having glucocerebrosidase activity and not having a sugar chain added thereto. is protein. According to this embodiment, a glycosylated protein having a sugar chain with a single structure and glucocerebrosidase activity is provided.
• glucose-cocerebrosidase activity means activity to hydrolyze glucocerebroside.
• the presence or absence of glucocerebrosidase activity is determined based on the presence or absence of enzymatic reactivity with respect to the synthetic substrate (p-nitrophenyl- ⁇ -D-glucopyranoside) described in the Examples section below.
• the specific activity of the protein having glucocerebrosidase activity to which no sugar chain has been added after the refolding treatment according to the present invention is, for example, 0.5 U/mg or more, preferably 0.6 U/mg or more. and more preferably 1.2 U/mg or more.
• the glycosylated protein of the present invention has a specific activity of 80% or more, preferably 100% or more, relative to the specific activity of the protein having glucocerebrosidase activity, which is not glycosylated. .
• the mature protein of glucocerebrosidase is a polypeptide consisting of 497 amino acid residues produced by cleaving the propeptide from the precursor protein consisting of 536 amino acid residues.
• Glucocerebrosidase biopharmaceuticals marketed for Gaucher disease include Cerezyme® (produced by Chinese Hamster Ovary (CHO) cells), VPRIV® (human fiber sarcoma cells (HT1080)), Elelyso® (produced by plant (carrot) cells).
• the amino acid sequence constituting the protein having no sugar chain and having glucocerebrosidase activity includes the amino acid sequence constituting the human wild-type GBA protein; Cerezyme (registered trademark); Amino acid sequences constituting VPRIV (registered trademark), Elelyso (registered trademark), etc.; synonymous with "homology” in the literature).
• amino acid sequences can be determined using analysis programs such as BLAST, FASTA, and CLUSTAL W. When using BLAST, use the program's default parameters.
• the "identity" of the amino acid sequences refers to aligning the two amino acid sequences so that the amino acid residues of the two amino acid sequences to be compared are matched as much as possible, and then determining the number of matched amino acid residues. is divided by the total number of amino acid residues and expressed as a percentage. In the above alignment, if necessary, gaps are inserted into one or both of the two sequences to be compared, and one inserted gap is counted as one amino acid residue to obtain the total number of amino acid residues. If the total number of amino acid residues determined in this way differs between the two sequences being compared, then the percent sequence identity is the total number of amino acid residues in the longer sequence and the number of matching amino acid residues. Calculated by dividing numbers.
• the protein having no sugar chain and having glucocerebrosidase activity is the amino acid sequence set forth in SEQ ID NO: 1 or 2 or an amino acid having 90% or more identity with them Contains arrays.
• the amino acid sequence described in SEQ ID NO: 1 corresponds to the amino acid sequence of selezyme (the amino acid at the position corresponding to position 495 is histidine (H), unlike the human wild-type GBA protein).
• the amino acid sequence is shown below, and the nucleotide sequence (including the termination codon) of the gene (cDNA) encoding the amino acid sequence is shown in SEQ ID NO:134.
• the gene encoding the amino acid sequence of SEQ ID NO: 1 is also simply referred to as "GBA gene".
• amino acid sequence set forth in SEQ ID NO: 2 corresponds to the amino acid sequence of biprib (the amino acid at the position corresponding to position 495 is arginine (R), unlike the human wild-type GBA protein).
• the amino acid sequence is shown below.
• the protein having no sugar chain and having glucocerebrosidase activity is represented by SEQ ID NO: 1 or 2.
• Amino acid sequences having at least one of the above amino acid substitutions include, for example, the amino acid sequences set forth in SEQ ID NOS: 16, 24, 28, 30, 37, 39, 41, 43-49, 136, 137, 141-145. .
• the protein having no sugar chain and having glucocerebrosidase activity according to the present invention is selected from the amino acid sequences set forth in SEQ ID NOs: 24, 30, 43, 136, 137, 141-145. including at least one
• the protein having no sugar chain and having glucocerebrosidase activity is the amino acid set forth in SEQ ID NO: 1 or 2.
• the protein having no sugar chain and having glucocerebrosidase activity according to the present invention is more preferably an amino acid having at least one of the following amino acid substitutions in the amino acid sequence of SEQ ID NO: 1 or 2:
• amino acids at the following positions are not substituted: In (a-2), the amino acid at the position corresponding to position 142 in (a-2), the amino acid at the position corresponding to position 144 in (a-2), the amino acid at the position corresponding to position 147 (a-2) In the amino acid (a-2) at the position corresponding to position 171, at the amino acid (a-2) at the position corresponding to position 347, at the amino acid (a-4) at the position corresponding to position 407, at position 248 In the amino acid at the corresponding position (a-9), the amino acid at the position corresponding to position 77 in (a-9), the amino acid at the position corresponding to position 290 in (a-9), the amino acid at the position corresponding to position 293 The amino acid at the position corresponding to position 333 in (a-9) The amino acid at the position corresponding to position 466 in (a-9).
• Amino acid sequences having at least one of the above amino acid substitutions include, for example, SEQ ID NOs: 3-5, 7, 9-30, 32, 33, 35, 37-39, 41-51, 136, 137, 141-145. and the amino acid sequence of
• the protein according to the invention is , 141-145.
• the protein having no sugar chain and having glucocerebrosidase activity according to the present invention is the amino acid of SEQ ID NO: 1 or 2
• the non-glycosylated and having glucocerebrosidase activity according to the present invention contains at least one amino acid sequence selected from the amino acid sequences set forth in SEQ ID NOs: 14, 17, 18 and 51. .
• a protein having no sugar chain and having glucocerebrosidase activity according to the present invention may consist of the amino acid sequence described above.
• the glycosylated protein of the present invention is obtained by adding a sugar chain having a single structure to a protein having glucocerebrosidase activity and not having a sugar chain added thereto.
• the number of amino acids to which sugar chains are added may be 1 or more, preferably 1-4.
• sugar chain means a compound in which one or more unit sugars (monosaccharides and derivatives thereof) are linked. When two or more unit sugars are connected, the unit sugars are bonded to each other by dehydration condensation due to glycosidic bonds.
• sugars include monosaccharides and polysaccharides contained in living organisms (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, complexes thereof and derivatives thereof); Degraded polysaccharides; sugar chains degraded or derived from complex biological substances such as glycoproteins, proteoglycans, glycosaminoglycans, and glycolipids.
• sugar chain having a single structure means that, when comparing the sugar chains to be added, the types of sugars constituting the sugar chain, the order of bonding, and the manner of bonding are the same as in the glycosylated protein. means that they are identical.
• the glycosylated protein of the present invention may have only one type of sugar chain added per site, but may also include proteins with two or more types of sugar chains added. .
• the sugar chain according to the present invention is not particularly limited as long as it does not eliminate the glucocerebrosidase activity of the glycosylated protein.
• the sugar chain may be a sugar chain that exists as a complex carbohydrate (glycopeptide or glycoprotein, proteoglycan, glycolipid, etc.) in vivo, or a sugar chain that does not exist as a complex carbohydrate in vivo. good.
• Sugar chains that exist as glycoconjugates in vivo include N-linked sugar chains, O-linked sugar chains, and the like.
• the glycosylated protein has one or more sugar chains having a single structure added thereto, and the sugar chain having a single structure is derived from a compound represented by the following formula 1: has the structure
• the sugar chain according to the present invention may be a sugar chain composed of the compound represented by Formula 1 above, or may be a sugar chain in which another sugar chain structure is bound to the compound represented by Formula 1.
• Sugar chains to which other sugar chain structures are bound include high-mannose type, complex type, hybrid type, and the like.
• the sugar chain according to the present invention consists of the compound represented by Formula 1 above.
• the amino acid to which the sugar chain is added is not particularly limited, and any amino acid can be used.
• the sugar chain may be directly bound to the amino acid, or may be attached via a linker.
• the sugar chain is attached via a linker.
• amino acids to which the sugar chain is added include cysteine, aspartic acid, glutamic acid, lysine, arginine, histidine, and tryptophan, from the viewpoint of ease of binding to the linker. , serine, threonine, tyrosine, asparagine, glutamine, and the like.
• the amino acid to which a sugar chain is added is preferably an amino acid selected from the group consisting of cysteine, asparagine, aspartic acid, glutamic acid, lysine, arginine, serine, threonine and glutamine, and more preferably. Cysteine.
• the linker is not particularly limited, and conventionally known ones can be used.
• the number of sugar chains linked to the linker is, for example, 1 or more, and It is preferably 1 to 3, more preferably 2 or 3, still more preferably 3, from the viewpoint of improving the amount of uptake.
• Examples in which three sugar chains are linked to a linker include a form in which a sugar chain is linked to each amino acid of a linker containing a tripeptide.
• the glycosylated protein of the present invention is not glycosylated, and a cysteine residue constituting a protein having glucocerebrosidase activity is glycosylated with a single-structure sugar chain. ing.
• the linker has a reactive functional group, preferably a maleimide structure, at its end.
• the protein having no sugar chain and having glucocerebrosidase activity has the amino acid sequence of SEQ ID NO: 1 or 2, wherein the above (1) to ( A protein having at least one amino acid substitution of 6) is preferred.
• the method for producing a peptide chain as a raw material for a protein having glucocerebrosidase activity and having no sugar chain according to the present invention is not particularly limited as long as a sugar chain is not added to the peptide chain. It may be a produced peptide chain or a peptide chain synthesized by organic synthesis. From the viewpoint of high productivity and low cost, the protein according to the present invention can preferably be derived from peptide chains produced by prokaryotes.
• prokaryotes examples include E. coli such as Escherichia coli, Bacillus such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida, and Rhizobium such as Rhizobium meliloti. Bacteria belonging to.
• the prokaryote used in the present invention is preferably E. coli.
• the method for producing a protein having glucocerebrosidase activity and not having a sugar chain includes introducing a vector containing a nucleic acid encoding the protein into a prokaryote, It includes producing a protein raw material in a prokaryote and subjecting the recovered protein raw material to a folding treatment.
• a vector containing a nucleic acid encoding the protein of the present invention is introduced into a prokaryote to produce a raw protein material. This makes it possible to obtain a raw protein material to which sugar chains have not been added.
• a method for producing a nucleic acid encoding a protein having glucocerebrosidase activity and having no added sugar chain according to the present invention and a vector containing the nucleic acid is not particularly limited, and conventionally known methods can be used. .
• vectors known vectors such as T vectors such as pTAKN-2 and plasmid vectors such as pET-21b(+) can be used.
• the method for introducing the vector into prokaryotes is not particularly limited, and conventionally known methods can be used as appropriate.
• Methods of introduction include a competent cell method, a conjugative transfer method, a calcium phosphate method, a lipofection method, an electroporation method and the like.
• the prokaryotic organism By culturing the prokaryotic organism into which the vector has been introduced, the prokaryotic organism can be made to produce a protein raw material. Cultivation of prokaryotes can be performed according to conventional methods used for the selected prokaryote.
• Prokaryotes are cultured under aerobic or anaerobic conditions, depending on the type of prokaryotes used.
• the prokaryotic culture may be subjected to shaking, aeration, or the like.
• the culture conditions (culture temperature, culture time, medium pH, etc.) are appropriately selected depending on the composition of the medium and the culture method, and are not particularly limited as long as the conditions allow prokaryotes to proliferate. can be selected as appropriate.
• the protein according to the present invention having no sugar chain and having glucocerebrosidase activity does not have a sugar chain due to post-translational modification, it is desirable that the protein is not post-translationally modified.
• prokaryotes are collected from the resulting culture by methods such as centrifugation and filtration, and the collected prokaryotes are subjected to mechanical methods such as beads or enzymatic methods. crush. After crushing, the insoluble fraction is collected and treated with a buffer containing a surfactant to recover the protein raw material.
• the collected protein raw material is subjected to folding treatment (refolding treatment including prior denaturation treatment is also acceptable).
• a buffer containing an oxidizing agent and a reducing agent oxidized glutathione/reduced glutathione, cystine/cysteine, cysteamine/cystamine, etc.
• oxidized glutathione/reduced glutathione, cystine/cysteine, cysteamine/cystamine, etc. is added to a liquid containing the recovered protein raw material, and the mixture is heated at about 20°C to about 20°C. It can be carried out by standing at about 30° C. for about 1 to 7 days. Further additives such as sucrose and glycerol can be added to the buffer.
• the recovered protein raw material may be subjected to denaturation (solubilization) treatment before folding treatment, if necessary.
• the denaturation treatment can be performed using a denaturant such as 6M guanidine hydrochloride and 8M urea. By applying the denaturation treatment, the recovered protein raw material can be brought into an unfolded state.
• the method for producing a sugar chain having a single structure according to the present invention is not particularly limited, and conventionally known methods can be used as appropriate.
• conventionally known methods can be used as appropriate.
• the methods described in WO03/008431, WO2004/058984, WO2004/058824, WO2004/070046, WO2007/011055, etc. can be used. can.
• cysteine which constitutes a protein having glucocerebrosidase activity and not having sugar chains added
• cysteine which constitutes a protein having glucocerebrosidase activity and not having sugar chains added
• sugar chain derivative (glycosylation reagent in the Examples) in which the sugar chain and the linker are bound is added to the glucocerebrosidase without the addition of a sugar chain. React with an active protein.
• a sugar chain derivative is, for example, a compound represented by Formula 1 in which the group bonded to the 1-position carbon of GalNac at the sugar chain terminal is replaced with a linker. Methods for producing linkers and sugar chain derivatives are not particularly limited, and conventionally known methods can be used.
• a sugar chain derivative and a protein having glucocerebrosidase activity to which no sugar chain has been added are reacted in a phosphate buffer at about 0°C to room temperature.
• the phosphate buffer may contain tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or the like to prevent dimer formation.
• TCEP tris(2-carboxyethyl)phosphine hydrochloride
• the final concentration of TCEP, etc. is, for example, 10 ⁇ M to 10 mM.
• the glycosylated protein can be obtained by purifying with HPLC.
• the glycosylated protein having glucocerebrosidase activity of the present invention has a sugar chain with a single structure added, unlike conventional glycosylated glucocerebrosidase proteins. Therefore, stable efficacy can be expressed. In addition, the stability at near neutral pH can be improved (see Examples). Therefore, it can be suitably used in the treatment of lysosomal diseases such as Gaucher disease.
• one embodiment of the present invention relates to compositions comprising said glycosylated proteins, preferably compositions comprising only one of said glycosylated proteins.
• the type and proportion of the glycosylated protein in the composition can be controlled.
• only one type of glycosylated protein in the composition means that the positions of amino acids to which sugar chains are added, the number of amino acids to which sugar chains are added, and the number of sugar chains to be added are the same. It means that some glycosylated protein is present in the composition.
• composition according to the present invention can have a constant quality, it is particularly suitable for uses such as pharmaceuticals and assays.
• the plasmid number and recombinant protein number are assigned the same number.
• GBA Glucocerebrosidase
• SEQ ID NO: 135 adds an initiation codon (atg) to the 5′-end of the codon encoding the mature GBA protein from which the signal peptide has been removed, and Modifications were made so that the sequence was optimized for the codon usage of E. coli (strain K-12).
• the synthesis of the GBA gene represented by SEQ ID NO: 135 was outsourced to Eurofins Genomics, Inc., and delivered in a state of being inserted into pTAKN-2 containing the ampicillin resistance gene.
• GBA gene-inserted plasmid In order to examine expression in E. coli, the GBA gene obtained above was inserted between the NdeI site and the His tag of the pET-21b(+) plasmid vector (Novagen). subcloned into. Specifically, PCR was performed using either pET-21b (+) or pTAKN-2 into which the GBA gene was inserted as a template, and linearized pET-21b (+) and the GBA gene (excluding the stop codon) were ) were obtained respectively.
• the PCR amplification product obtained above was treated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) (cleavage and ligation with restriction enzyme DpnI), and the pET-21b(+) plasmid into which the GBA gene was inserted A vector (referred to herein as "H495 type") was obtained.
• the GBA gene inserted into the plasmid vector encodes the amino acid sequence set forth in SEQ ID NO:1.
• the resulting PCR amplified product (linearized plasmid) was fused with T4 Polynucleotide Kinase (Toyobo Co., Ltd.) and Ligation high Ver. 2 (Toyobo Co., Ltd.) by self-ligation to obtain a plasmid into which the modified GBA gene was inserted (Table 5).
• T4 Polynucleotide Kinase Toyobo Co., Ltd.
• Ligation high Ver. 2 Toyobo Co., Ltd.
• E. coli competent cells ECOS competent E. coli BL21 (DE3) (Nippon Gene Co., Ltd.)
• Various recombinant E. coli strains were constructed that were transformed and carried plasmid vectors into which the GBA gene or modified GBA gene had been inserted.
• a single colony grown on LB agar medium (containing ampicillin at a concentration of 100 mg/L) was added to 4 mL of LB liquid medium (containing ampicillin at a concentration of 100 mg/L) in a test tube.
• the cells were inoculated and cultured with shaking at 300 rpm and 30° C. overnight to obtain a preculture solution.
• the culture medium is centrifuged at 6,000 x g for 10 minutes at 4°C, the supernatant is discarded, and the precipitate is suspended using buffer A (see Table 4 below for composition). let me After that, the mixture was centrifuged again at 6,000 ⁇ g and 4° C. for 10 minutes, and after discarding the supernatant, a precipitate of recombinant E. coli was obtained (then it was frozen and stored at ⁇ 80° C.).
• the denominator of 1.7 is the extinction coefficient calculated based on the amino acid sequence information.
• Incubation was started by standing at 25°C from the time of dilution, samples were collected 7 days after the start of incubation, and enzyme activity was measured by the following method.
• GBA Glucocerebrosidase
• Glc-Cer glucocerebroside; glycolipid
• pNPG synthetic substrate p-nitrophenyl- ⁇ -D-glucopyranoside
• the GBA protein having the amino acid sequence of SEQ ID NO: 1 (herein referred to as "H495 type protein") produced in E.
• C342 is an amino acid residue necessary for enzymatic activity (THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 7, pp. 4242-4253, February 17, 2006). However, it was found that the activity was maintained when the serine was substituted.
• Each recombinant GBA protein was obtained from each recombinant E. coli strain carrying the plasmids listed in Table 6 by the same method as 2-1 to 2-3 above.
• Liquid A buffer C (see Table 9 below for the composition) and liquid B: ethanol were used as solutions, and the active fraction eluted at 40% B was collected. The recovered solution was concentrated with Amicon Ultra-15, 3 kDa (Merck) and then lyophilized.
• DiMan-Asn 1 (119.8 mg, MW: 1024.9, 117 ⁇ mol) was suspended in DMF, and N-Succinimidyl 3-Maleimidopropionate (78.0 mg, MW: 266.2, 293 ⁇ mol, 2.5 eq, Wako, product code : QA-2328) and diisopropylethylamine (DIPEA, 102 ⁇ L, 585 ⁇ mol, 5.0 eq, nacalai tesque, product code: 14014-84) were added and allowed to react at room temperature for 24 hours. After 24 hours from the initiation of the reaction, the reaction mixture was dropped into ethyl acetate to precipitate. The precipitate was washed twice with ethyl acetate and dried. Purification by HPLC gave Di-Man-Asn-MAL 2 (92.0 mg, MW: 1176.05, 78.2 ⁇ mol, 67%).
• S242C was confirmed to have a reduced enzymatic activity due to modification.
• T61C, P98C, Q143C, K224C, K321C and T407C are suitable as modification positions.
• Cerezyme registered trademark
• purified recombinant GBA proteins No. 167 and No. 178
• Table 11 shows the results.
• Cell uptake test (Test example 1) NR8383 cells (purchased from ATCC) were cultured in Ham's F12K medium (containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% heat-inactivated fetal bovine serum).
• a test solution was prepared in a 1.5 mL tube using Ham's F12K medium so as to have the following composition.
• a glycosylated active glucocerebrosidase variant No. 178-G1 or No. 178-G3 was used.
• Test example 2 A recombinant GBA protein (No. 178) or a glycosylated active glucocerebrosidase variant (No. 178-G3) was used as a sample, and the test solution was prepared so as to have the following composition. The specific activity was calculated as in Example 1. Table 15 shows the results.
• Test example 3 Sugar chain-modified active glucocerebrosidase variant (No. 178-G3 or No. 234-G3) was used as a sample, a test solution was prepared to have the following composition, and incubated at 37°C for 1.5 minutes. The specific activity was calculated in the same manner as in Test Example 1, except that it was carried out for a period of time. The results are shown in Table 16.

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