CN101410408A - A process for concentration of a polypeptide - Google Patents

Abstract

The present invention comprises a method of concentrating a composition comprising a polypeptide of interest and the use of such a concentrated composition for the treatment of diseases in mammals, in particular by subcutaneous injection.

Description

Be used for concentrating the method for polypeptide

Invention field

The present invention relates to be used for concentrate the method for polypeptide of interest, relate to and comprise the composition that concentrates polypeptide of interest as being used for the purposes of hypodermic medicine and relating to and comprise the composition of 10mg/ml polypeptide of interest at least.

Background of invention

Some polypeptide are used as medicine to prevent and/or treat some disease.The ability of subcutaneous injection medicine is a kind of advantage, because this makes the patient oneself use this medicine easily.

Because to having physiology restriction (physiological restrain) by the much volumes of subcutaneous injection; therefore for the medicine of subcutaneous administration; advantageously they can obtain with high density, accept enough drug and/or avoid multiple subcutaneous injections to guarantee the patient.

WO 99/37325 discloses treatment and has prevented by the activity shortage of the enzyme that belongs to the protoheme biosynthetic pathway or the method for defective associated diseases.WO 03/002731 discloses and has been used for the method for purification of Recombinant porphobilinogen deaminase in commercial quantity, and relates to the purposes that described purified product is used to prepare medicine.Similarly, WO 02/099092 and WO 2005/094874 provide lysosome alpha-Mannosidase and therapeutic use thereof.At last, WO 2005/073367 is provided for method and the purposes of this enzyme in the treatment metachromatic leukodystrophy of purifying aryl sulphatase A.

The present invention relates to be used for concentrate the method for polypeptide of interest, and relate to and comprise the composition that concentrates polypeptide of interest and be used for the purposes of subcutaneous injection to mammiferous medicine in preparation.

Summary of the invention

The present invention relates to the concentrated method for compositions that comprises polypeptide of interest in one aspect, and it comprises:

A) centrifugal and/or filtration comprises the composition of polypeptide of interest,

B) concentrate supernatant liquor or the retentate that obtains from step a) respectively.

In yet another aspect, the present invention relates to comprise the composition of 10mg/ml polypeptide of interest at least.

In yet another aspect, the composition that the present invention relates to comprise the 75-250mg/ml polypeptide of interest is used for the purposes of subcutaneous injection to mammiferous medicine in preparation.

In yet another aspect, the present invention relates to treat the method for Mammals acute intermittent porphyria, it comprises that subcutaneous injection contains the composition of 500-300mg/ml PBGD.

In yet another aspect, the present invention relates to treat the method for Mammals metachromatic leukodystrophy, it comprises that subcutaneous injection contains the composition of 50-300mg/ml aryl sulphatase A (Aryl sulfatase A).

In yet another aspect, the present invention relates to treat the method for Mammals lysosomal storage disease α-mannosidosis, the composition of subcutaneous injection 50-300mg/ml lysosome alpha-Mannosidase.

In yet another aspect, the present invention relates to treat the method for Mammals Krabbe disease, it comprises that subcutaneous injection contains the composition of 50-300mg/ml galactocerebrosidase (galactosycerebrosidase).

Definition

Be purpose of the present invention, the calculating that the comparison of sequence and homology are kept the score can use the complete Smith-Waterman comparison method that can be used for protein comparison and DNA comparison to carry out.Acquiescence is kept the score, and matrix B LOSUM50 and identity matrix are respectively applied for the protein comparison and DNA compares.The point penalty of first residue is-12 for protein and is-16 for DNA in the breach (gap), and the point penalty of extra residue is-2 for protein and is-4 for DNA in the breach.Comparison can be used FASTA software package v20u6 version (W.R.Pearson and D.J.Lipman (1988), " the improvement instrument that is used for biological sequence analysis ", periodical (PNAS) 85:2444-2448 of institute of NAS and W.R.Pearson (1990) " with the quick and responsive sequence alignment of FASTP and FASTA ", method in the zymetology (Methods in Enzymology) 183:63-98) is carried out.

The multiple ratio of protein sequence is to using " ClustalW " (Thompson, J.D., Higgins, D.G and Gibson, TJ. (1994) CLUSTAL W: the susceptibility of selecting to improve carrying out property multiple sequence comparison method by sequence weighting, location specific breach point penalty and weight matrix.Nucleic acids research (NucleicAcids Research) 22:4673-4680) carries out.The multiple ratio of dna sequence dna is to using the protein comparison result as template, to replace amino acid from the corresponding codon of dna sequence dna and to carry out.

In the context of the present invention, term " E.C. " (enzyme classification) refers to the enzyme classification system of international endorsement, and international biological chemistry and NK of molecular biology federation are recommended, academic press (AcademicPress), Inc.

Used term " source " will be interpreted as and mean from wherein obtaining the biology of described sequence under the context environmental of aminoacid sequence (for example protein) or nucleotide sequence.This sequence can use the well-known gene engineering method of those skilled in the art by another kind of biological the expression.This sequence also comprises by the sequence of chemosynthesis.In addition, this sequence can comprise trickle change such as codon optimized, i.e. the change that does not influence aminoacid sequence in the nucleotide sequence.

Detailed Description Of The Invention

Polypeptide of interest

Polypeptide of the present invention especially can be hormone or hormone variant (hormone variant), enzyme, acceptor or its part, antibody or its part, allergen or reporter molecules.Polypeptide of interest especially can be selected from one of six main groupings of enzyme, for example enzyme in oxydo-reductase (E.C.1), transferring enzyme (E.C.2), lytic enzyme (E.C.3), lyase (E.C.4), isomerase (E.C.5) or the ligase enzyme (E.C.6).Aspect more specifically, polypeptide of interest can be an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, cellobiohydrolase, chitinase, at, Maltose 4-glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase enzymes, glucoamylase, alpha-glucosidase, beta-glucosidase enzyme, saccharase, laccase, lipase, mannosidase, dextranase (mutanase), oxydase, pectin degrading enzyme, peroxidase, Phospholipid hydrolase, phytase, polyphenoloxidase, proteolytic ferment, rnase, trans-glutaminases, zytase or xylobiase.

Polypeptide of interest especially can be the polypeptide of useful as drug.

The example of suitable polypeptide of interest includes but not limited to be selected from one of them enzyme of porphobilinogen deaminase, aryl sulphatase, alpha-Mannosidase and galactocerebrosidase.

In principle, can handle according to the inventive method from the obtainable polypeptide of interest in any source.

In embodiment, polypeptide of interest can be the people source.Especially using polypeptide of interest to make under the situation of the medicine that will be applied to the people, described polypeptide can be the people source, because this can make undesirable allergic risk minimization.Term " human origin " for example comprises the natural variation because of the human polypeptides due to the polymorphism in the context of the invention.

Polypeptide of interest especially can be used as recombinant protein production, and the nucleotide sequence of the polypeptide of interest of promptly encoding can import the cell that is used to express polypeptide of interest.Recombinant expressed can be homologous or allogenic, and promptly polypeptide of interest can be in the cell of natural this polypeptide of expression be expressed (homology expression) or it can be by the cell expressing (heterology expression) of not natural this polypeptide of expression natively.

The reorganization polypeptide of interest can produce any cell expressing of specific polypeptide of interest by being suitable for recombinating.The example of suitable cell includes but not limited to prokaryotic cell prokaryocyte, as intestinal bacteria (E.coli) cell or genus bacillus (Bacillus) cell.Suitable eukaryotic example includes but not limited to yeast cell or as the mammalian cell of Chinese hamster ovary cell (CHO).Alternatively, it can be people's cell.

The suitable host cell that is used to express glycosylated polypeptides derives from multicellular organism (multicellularorganism).The example of invertebral zooblast comprises vegetable cell and insect cell.Yet host cell also can be a vertebrate cells, and the vertebrate cells propagation of cultivating (tissue culture) has been become ordinary method.

The polypeptide that term " recombinant polypeptide " or " reorganization polypeptide of interest " refer in this article to recombinate and produce.

The function equivalent part or the analogue that specific polypeptide of interest appellation are also comprised in the context of the present invention polypeptide of interest.For example if polypeptide of interest is an enzyme, then the function equivalent of this enzyme part can be the structural domain like this or the subsequence (subsequence) of this enzyme, and it comprises the essential catalytic site that can produce the enzymic activity substantially the same with the gene of total length enzyme or selectable coding catalyzer.Term " substantially the same enzymic activity " refers to have at least 50%, preferred at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% active equivalence part of natural enzyme or analogue.The example of the zymetology equivalence analogue of enzyme can be to comprise being in the fusion rotein that this enzyme catalysis site under the functional form is arranged, but it also can be the homology variant that derives from this enzyme of another species.In addition, the complete artificial molecule of the specificity enzymic activity of simulation relevant enzyme also constitutes " zymetology equivalence analogue ".

Usually, the technician can be designed for the suitable assay method of measuring enzymic activity easily.Yet for PBGD, suitable assay method reaches in WO 03/002731, in embodiment 2 and describes in the application's experimental section.Except that its natural substrate, also can the catalysis synthetic property chromogenic substrate of aryl sulphatase is right-hydrolysis of Nitrocatechol sulfuric ester (pNCS).Right-Nitrocatechol (pNC) product is absorbed in the light on the 515nm.The assay method that is used to measure arylsulfatase activity is at WO 2005/073367 and at Fluharty etc. 1978, and Enzymology method is learned among (Meth.Enzymol) .50:537-47 and described in detail.For LAMAN, suitable enzyme assay method is open in WO 02/099092.

Porphobilinogen deaminase

In one embodiment, polypeptide of interest of the present invention can be porphobilinogen deaminase (being called porphobilinogen ammonia-lyase (polymerization) again) be E.C.4.3.1.8. (

1937, J.Acta.Med.Scand.Supp1.8).Porphobilinogen deaminase is the 3rd enzyme in the protoheme biosynthetic pathway.E.C.4.3.1.8 is renamed as E.C.2.5.1.61, so porphobilinogen deaminase (PBGD) is positioned at this E.C. numbering down now.

Porphobilinogen deaminase catalysis is so reacted: 4 porphobilinogen+H ₂O=methylol bilane+4 NH ₃

PBDG and acute intermittent porphyria (AIP) have important relationship, and wherein acute intermittent porphyria is to lack autosomal dominant disorder (other details of relevant this disease is seen WO01/07065) due to (active reduce by 50%) because of PBDG among the mankind.

Porphobilinogen deaminase abbreviates PBGD as and this two terms can use in the context of the present invention each other with exchanging.

Recombinant expressed for PBGD, host cell especially can be yeast cell or Bacillus coli cells.

For the detailed example that makes up recombinant Bacillus coli cells, with reference to the embodiment 1 of WO01/07065 and for making up reorganization HeLa cell and the reorganization NIH 3T3 cell that to express mouse PBGD, with reference to the embodiment 6 of WO01/07065.

Term " reorganization porphobilinogen deaminase (rPBGD) " refers to the PBGD that recombinates and produce in this article.Hereinafter, the people PBGD form of this kind of enzyme and reorganization thereof will be called " PBGD " and " rhPBGD " respectively.The zymetology equivalence part or the analogue that in this term, also comprise PBGD.An example of the zymetology equivalence part of this enzyme can be the structural domain like this or the subsequence of this enzyme, and it comprises the essential catalytic site that can produce the enzymic activity substantially the same with the gene of total length enzyme or selectable coding catalyzer.Term " substantially the same enzymic activity " refers to the equivalence part like this or the analogue of this enzyme, and it has measured natural human rhPBGD activity at least 50%, preferred at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% rhPBGD activation measurement of describing in the embodiment 2 of WO 03/002731.The example of the zymetology equivalence analogue of this enzyme can be to comprise being in the fusion rotein that this enzyme catalysis site under the functional form is arranged, but it also can be the homology variant that derives from this enzyme of another species.In addition, the complete artificial property molecule of the specificity enzymic activity of simulation relevant enzyme also constitutes " zymetology equivalence analogue ".

The example of the PBGD that can use in the present invention be included in this please sequence 1-10 shown in or have any one of those PBGD of Genebank numbering X04217, X04808 or M95623.

In yet another embodiment of the present invention, polypeptide of interest can be aryl sulphatase A (Arylsulfatase A).

The following reaction of aryl sulphatase A catalysis: 3-cerebroside sulfate+H ₂O=cerebroside+vitriol (sulphate).

From the multiple source that comprises people's liver, placenta and urine purifying ASA.ASA is the acid glycoprotein with low iso-electric point.Greater than pH 6.5, this enzyme exists as the dimer of the about 110kDa of molecular weight.The polymerization of pH-dependency takes place in ASA, forms eight aggressiveness on pH 4.5.In people urine, this enzyme by 63 and two subunits inequality of 54kDa constitute.From people's liver, placenta and inoblast the ASA of purifying also by the different slightly molecular weight of size 55 and 64kDa between two subunits changing constitute.As other lysosomal enzyme, ASA is synthetic as the glycosylation precursor on film binding ribosomal body.This glycosylation precursor passes endoplasmic reticulum and golgi body subsequently, the oligosaccharides of the N-of glycosylation precursor connection therein obtains processing, form phosphorylation and Sulfated complexity oligosaccharides (biological chemistry and Acta Biophysica Sinica (Biochim Biophys Acta) .1985 such as Waheed A, 847,53-61, biological chemistry and biophysical studies communication (Biochem Biophys Res Commun) .1987 such as Braulke T, 143,178-185).In the normal inoblast of cultivating, produce the precursor polypeptide of 62kDa, this precursor polypeptide is by the effect of Man-6-P receptors bind and transposition (journal of biological chemistry (J Biol Chem) .1990 such as Braulke T, 265,6650-6655) to acid prelysosome endosome (cytobiology such as Kelly BM Europe magazine (EurJ Cell Bio) 1.1989,48,71-78).

Aryl sulphatase A especially can be the people source.The length of people ASA signal peptide (18 amino acid) is based on the specificity processing site of consensus sequence and signal sequence.Therefore, should produce the people ASA of mature form all carrying out afterwards at residue numbering 18 (Ala) in the cells to cutting from the signal peptide of derivation people ASAcDNA (EMBL GenBank accession number J04593 and X521151).Hereinafter, reorganization aryl sulphatase A will be abbreviated as rASA, and the aryl sulphatase A of mature form (the people ASA that comprises mature form) will be called " mASA " and sophisticated recombinant human ASA will be called " mrhASA ".

A kind of protein modification is determined in two kinds of eucaryon sulfatases (ASA and ARB (ASB)), and one of these two kinds of eucaryon sulfatases are volvox (Volvox carteri) (cell (Cell) .1995 such as Schmidt B from green alga, 82,271-278, biological chemistries such as Selmer T Europe magazine (EurJ Biochem) .1996,238,341-345).This modification cause in known sulfatase conserved cysteine residue change into 2-amino-3-oxo propionic acid residue (Cell.1995 such as Schmidt B, 82,271-278).This amino acid derivatives also is identified as C*-formylglycine (FGly).In ASA that derives from the MSD cell and ASB, the Cys-69 residue is kept.Therefore, the conversion that proposes Cys-69 to FGly-69 is essential for producing catalytic activity ASA and ASB, and the shortage of this protein modification is the cause of disease of MSD.Cys-69 is with reference to having 18 residue signal propeptide ASA.In mASA, mentioned cysteine residues is Cys-51.Verified one the 16 residue line style sequence of surrounding Cys-51 in mASA of other research is enough to instruct this conversion and this protein modification to occur in common translation protein translocation to endoplasmic reticulum or in its late period, this moment, polypeptide still was not folded into its natural structure (periodical (Proc Natl Acad Sci) .1997 of institute of NAS such as Dierks T, 94,11963-1196, Wittke, D. etc. (2004), neuropathology journal (Acta Neuropathol). (Berl.), 108,261-271).

The ASA of various ways confirms on from the electrophoresis of the enzyme prepared product of the inoblast of people's urine, white corpuscle, thrombocyte, cultivation and liver and isoelectric focusing electrophoresis.Reduced the complicacy of molecular size and electrophoretic pattern with endoglycosidase H, sialidase and alkaline phosphatase treatment, this shows the sugared content of a large amount of electric charge unhomogeneities of ASA owing to this enzyme.

Aryl sulphatase A can stride across the aryl sulphatase A form of hemato encephalic barrier and/or possess the rASA form that is used to enter the specificity label of target cell in the brain.Especially, it can be the rASA that obtains efficient endocytosis in vivo by the Man-6-P approach.

Therefore, ASA especially can with covalently be connected as so-called label, peptide or the protein of carrier or as the toxin of carrier, wherein said carrier can increase and/or promote the ASA transportation to pass hemato encephalic barrier usually and/or stride across cytolemma (Schwarze etc., cytobiology trend (Trends Cell Biol) .2000; 10 (7): 290-295; Lindgren etc., pharmacology science trend (Trends Pharmacol.Sci) .2000; 21 (3): 99-103).The ASA molecule that contains this type of peptide sequence can produce by expression technology.The protein transduction process is not cell type-specific, and the mechanism that this process takes place do not throw a flood of light on, yet, it is believed that the film disturbance of dependent certain type of this process factor receptor and penetrate process and take place.The part of molecule is separated folded state may promote this process, but optional.

The example of appropriate label includes but not limited to the Man-6-P label.

Peptide or proteinic example as carrier include but not limited to so-called protein transduction domains.The example of suitable protein transduction domains includes but not limited to those protein transduction domainses of mentioning in WO 2005/073367, wherein said document is by with reference to incorporating in this article.Therefore, protein transduction domains can be from the proteic 11 residue basic peptide-YGRKKRRQRRR of HIV TAT (Schwarze etc., Trends Cell Biol.2000; 10 (7): 290-295), sequence given synthesized form-YARAAARQARA (Ho etc., cancer research (Cancer Res) .2001 of the TAT of more alpha-helixs and amphoteric character; 61 (2): 474-477), the synthetic property leading peptide that constitutes by poly--R or with the mixture of the alkalescence-R of other amino acid combination and-K residue and based on partly peptide of the hydrophobicity signal sequence of integrating element or Ka Boqi sarcoma FGF from β-3 (biopolymer (Biopolymers) 2001 such as Dunican; 60 (1): 45-60).

Include but not limited to those toxin of mentioning as the example of the suitable toxin of carrier in WO 2005/073367, wherein said document is by with reference to incorporating in this article.

ASA especially can comprise such nucleotide sequence, its coding:

(a) aminoacid sequence of the SEQ ID NO:2 among the WO 2005/073367;

(b) in the part of sequence described in (a), this part and recombinant human aryl sulphatase A are the zymetology equivalences;

(c) aminoacid sequence analogue, its with (a) or the arbitrary sequence (b) have at least 75% sequence identity, and meanwhile comprise with recombinant human aryl sulphatase A be the aminoacid sequence of zymetology equivalence.

In the context of this article, the part of aminoacid sequence or aminoacid sequence is when measuring the active assay method of aryl sulphatase A and measure in being used for described in the embodiment 1 of WO2005/073367, can 37 ℃ with the polypeptide of a certain amount of aryl sulphatase A of the corresponding speed hydrolysis of the single-minded activity substrate pNCS of 20U/mg polypeptide at least (preferred 50U/mg polypeptide), and/or be when in the assay method described in the embodiment 2 of WO 2005/073367, analyzing by the incubation on dosage level 25mU/ml, the polypeptide that is loaded on the fibroblastic mark aryl sulphatase of MLD A substrate fx.14C palmityl sulfatide that can hydrolysis at least 40%.

ASA can especially comprise at another embodiment:

(a) nucleotide sequence of the SEQ ID NO:1 among the WO 2005/073367;

(b) in the part of sequence described in (a), this part coding is the aminoacid sequence of zymetology equivalence with recombinant human aryl sulphatase A;

(c) nucleotide sequence analogue, its with (a) or the arbitrary sequence (b) have at least 75% sequence identity and meanwhile coding be the aminoacid sequence of zymetology equivalence with recombinant human aryl sulphatase A.

Preferably the sequence identity degree between the SEQ ID of nucleotide sequence mentioned above and WO 2005/073367 NO:1 be at least 80%, as at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.Can be on an equal basis preferably be at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% by the sequence identity degree between the SEQ ID NO:2 of the aminoacid sequence of nucleic acid sequence encoding mentioned above and WO 2005/073367.

Be purpose of the present invention, preferred aryl groups sulfatase A is a recombinase, people's aryl sulphatase A (rhASA) of preferred especially reorganization.

Preferably in mammalian cell or clone, produce rASA, and the sugar form of described mammalian cell or clone generation rASA, described sugar form is able to efficient endocytosis by the Man-6-P receptor pathway in vivo.Particularly, the preferred sugar form of rASA comprises the Man-6-P of a certain amount of exposure, and this impels rASA to pass through the efficient endocytosis of Man-6-P approach in vivo.

In embodiment, the rASA sugar form that is produced is one of at least similar to the sugar form that produced in the Chinese hamster ovary celI.

The posttranslational modification of the cysteine residues in the position 51 that becomes acquaintance's aryl sulphatase A is relevant with the activity of this enzyme.Therefore in the preferred embodiment of the present invention, the generation of aryl sulphatase A or its equivalent is taking place with such speed and under condition like this, its generation comprises the product of this enzyme isoform, wherein is converted to the corresponding formylglycine of Fgly-51 with the SEQ ID NO:3 of WO 2005/073367 with the corresponding amino acid of Cys-69 of the SEQ ID NO:2 of WO 2005/073367 in described isoform.Behind the SEQ ID NO:4 of WO 2005/073367 representative excision 18 amino acid signal peptides and the one-tenth acquaintance aryl sulphatase A before modifying C-51.

Therefore in yet another embodiment of the present invention, ASA or its enzyme equivalent can be selected from

(a) aminoacid sequence of the SEQ ID NO:3 among the WO 2005/073367;

(b) part of sequence in (a), this part and recombinant human aryl sulphatase A are the zymetology equivalences;

(c) aminoacid sequence analogue, its with (a) or the arbitrary sequence (b) has at least 75% sequence identity and be the zymetology equivalence with recombinant human aryl sulphatase A meanwhile.

Can be preferably sequence identity degree between the SEQ ID NO:4 of the SEQ ID NO:3 of enzyme that produces according to the present invention and WO 2005/073367 or WO 2005/073367 be at least 80%, as at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.

Because the biologic activity of body endoenzyme and effect need be best, thereby if the enzyme of capacity has obtained aforesaid glycosylation pattern and be subjected to posttranslational modification on position 51, then this is favourable.Therefore at least 50%, 60%, 70%, 80%, 90%, 95% or 98% ASA of the present invention can be sugar form/isoform mentioned above.

ASA of the present invention can differ from the rASA according to 2005/073367 SEQ ID NO:3 with regard to its structure.It is identical with corresponding sequence among the SEQ ID NO:3 or have the sequence identity of high level advantageously to surround the sequence of Cys-51.Therefore, the 20 amino acid line style sequences of surrounding Cys-51 in can preferred aryl groups sulfatase A as 19,18,17,16,15,14,13,12,11,10,9,8,7,6,5 or 4 amino-acid residues identical with corresponding sequence among 2005/073367 the SEQ ID NO:3 or with as described in corresponding sequence at least 90% identical, identical as 95%, 96%, 97%, 98% or 99%.Because the activity form of rASA is eight aggressiveness in the lysosome, ASA of the present invention especially can be as eight aggressiveness or be assembled into the rASA of eight aggressiveness under physiological condition.

The enzymic activity of ASA (it should be understood to the catalytic activity of rASA) can be measured in such enzyme assay, and wherein said enzyme assay is based on detecting substrate or the rASA that produces the substrate that can detect end product being mediated the property hydrolysis.Aspect preferred, this assay method based on to have end product right-the synthetic property chromogenic substrate of Nitrocatechol (pNC) is right-hydrolysis of Nitrocatechol sulfuric ester (pNCS), wherein said right-Nitrocatechol is absorbed in the light on the 515nm.

The lysosome alpha-Mannosidase

In another embodiment, polypeptide of interest can be lysosome alpha-Mannosidase (LAMAN).The lysosome alpha-Mannosidase belongs to EC 3.2.1.24 and is to connect between the orderly degradative phase of glycoprotein the exoglycosidase (Aronson and Kuranda FASEB magazine (FASEBJ) 3:2615-2622.1989) of terminal irreducibility α-D-mannose residue from non reducing end hydrolyzing alpha-D-mannoside at N-.In the context of the present invention, term lysosome alpha-Mannosidase can use with phrase LAMAN exchange ground.

LAMAN of the present invention especially can be the people source.People source enzymic synthesis is inferred 1011 the amino acid whose single polypeptides that contain of signal peptide as having one 49 residue, wherein said infer signal peptide be processed into 15kD, 42kD and 70kD 3 kinds of main glycopeptides (human molecular genetics (Hum.Mol.Genet) .6 such as Nilssen, 717-726.1997).

The gene of coding LAMAN (MANB) is positioned at the 19th karyomit(e) (19cen-q12) karyomit(e) (Chromosoma) 95:8-12.1987 such as () Kaneda.MANB is made of 24 exons, covers 21.5kb (GenBank accession number U60885-U60899; Genomics such as Riise (Genomics) 42:200-207.1997).Nearly 3,500 Nucleotide of LAMAN transcript (nt) also contain coding 1,011 amino acid whose open reading-frame (ORF) (GenBank U60266.1).

In three pieces of papers, delivered the clone of people cDNA of coding LAMAN and order-checking (Hum.Mol.Genet.6 such as Nilssen, 717-726.1997; J.Biol.Chem.271 such as Liao, 28348-28358.1996; Biochem.Biophys.Res.Commun.200 such as Nebes, 239-245.1994).Making us curious is that these three kinds of sequences are inequality.When with the sequence (accession number U60266.1) of Nilssen etc. relatively the time, Liao etc. and Nebes etc. have found and have caused from Xie Ansuan to the aspartic acid metathetical that TA to AT changes on position 1670 and 1671.

In most preferred embodiments, lysosome α mannosidase comprises the aminoacid sequence of the SEQ ID NO.:1 of WO 2005/094874.

Be in practicality and economic cause, preferably be recombinantly produced LAMAN of the present invention.By recombinant production, also can obtain the prepared product of this enzyme, wherein most enzyme contains Man-6-P.Recombinant production can realize by the nucleotide sequence transfectional cell of sequence that use comprises the SEQ ID NO.:2 of WO 2005/094874.

Alpha-Mannosidase preferably produces in mammal cell line system, because this will be created in the glycosylation form of guaranteeing the efficient picked-up of receptor-mediated property in the cell of health internal organs for example.Especially, have been found that the generation of enzyme in CHO, COS or bhk cell guarantee to make the enzyme post transcriptional modificaiton fully by adding the Man-6-P residue.In addition, obtained correct sialylated form.Known to stop because of the liver picked-up due to the galactose residue that exposes, correct sialylated be important.

In addition preferred embodiment in, mammal cell line system thus be selected from CHO, COS cell or bhk cell (journal of biological chemistry (J Biol Chem) .1989 such as Stein, 264,1252-1259).Preferably the mammal cell line system is human fibroblasts's clone.

In most preferred embodiments, the mammal cell line system is a Chinese hamster ovary celI system.

In another embodiment, the lysosome alpha-Mannosidase can be the prepared product like this of lysosome alpha-Mannosidase, and the part of wherein said prepared product constitutes by having the lysosome α mannosidase that carries the one or more N-connection of seminose 6-phosphate oligosaccharides.

A part of prepared product of further preferably described lysosome alpha-Mannosidase can combine with seminose 6-phosphate acceptors.

The ability of this enzyme and Man-6-P receptors bind can be measured in the external test method described in the embodiment 1 of WO 2005/094874.This enzyme provides tolerance to this enzyme and Man-6-P receptor binding capacity to the combination of the affine 300Matrix of MPR.In the preferred embodiment of the present invention, enzyme is to the external generation of being combined in of Man-6-P acceptor.

In the preferred embodiment of the present invention, described part is corresponding to the 1-75% activity of lysosome alpha-Mannosidase prepared product, as 2-70%, as 5-60%, as 10-50%, as 15-45%, as 20-40%, as 30-35%.

Therefore, preferred lysosome alpha-Mannosidase has such seminose 6-phosphoric acid residue content, and it allows the enzyme of 2-100%, 5-95%, 10-90%, 20-80%, 30-70% or 40-60% amount to combine with Man-6-P-acceptor matrix generation seminose 6-phosphoric acid dependency.At present, phosphorylation degree in several batches enzyme, analyzed and usually the enzyme of 30-45% be phosphorylation and in conjunction with affinity matrix.

The part that further preferably occupies described lysosome alpha-Mannosidase amount 2-100%, 5-90%, 10-80%, 20-75%, 30-70%, 35-65% or 40-60% combines with high-affinity with the Man-6-P-acceptor.In theory, two seminose 6-phosphate groups must be on the position mutually near in case this enzyme with high-affinity in conjunction with the Man-6-P-acceptor.Nearest observations shows that the distance between the phosphorylation mannose residue must be 40 Or shorter, so that obtain the high-affinity combination.In the people's lysosome alpha-Mannosidase according to the SEQ IDNO:1 of WO 2005/094874, two seminose 6-phosphoric acid residues can be arranged in the asparagine residue place of position 367 and 766.Therefore, preferred medicine of the present invention comprises such lysosome alpha-Mannosidase, and its part is all carried seminose 6-phosphate group at these two asparagine residue places.

Preferably, alpha-Mannosidase produces by recombinant technology.In another embodiment, alpha-Mannosidase comes from people (hLAMAN) and still is more preferably sophisticated people's alpha-Mannosidase (mhLAMAN) or its fragment.This fragment can be modified, yet the reactive site of enzyme should keep.

Be expected in the prepared product of alpha-Mannosidase of the present invention, a part of portion of this enzyme is the precursor forms representative of enzyme thus, and other parts represent about 55 and the protease cracking form processing of 70kDa..

Galactocerebrosidase

In another embodiment, polypeptide of interest can be a galactocerebrosidase, and it can be abbreviated as GALC.Galactocerebrosidase belongs to E.C.3.1.6.46 and is the enzyme that can react below the catalysis: D-galactosyl-N-acyl sphingosine+H ₂O=D-semi-lactosi+N-acyl sphingosine, so for example degraded of galactolipid in the myelin of GALC catalysis.

The GALC enzyme that derives from the people is the glycosylation lysosomal enzyme that comprises 643 amino acid and have the 72.8kDa molecular weight.GALC of the present invention especially can be the people source.In another embodiment, GALC can express with recombinating in the host cell that preamble is mentioned.The host cell that is used for recombinant expressed GALC especially can be a Chinese hamster ovary celI.

Describe and in claims in invention, with reference to following amino acid sequences and nucleotide sequence:

Sequence description	The recognition sequence symbol
		PBGD encoding sequence 1	SEQ ID NO.：1
PBGD encoding sequence 2	SEQ ID NO.：2
		PBGD encoding sequence 3	SEQ ID NO.：3
PBGD encoding sequence 4	SEQ ID NO.：4
		PBGD encoding sequence 5	SEQ ID NO.：5
PBGD encoding sequence 6	SEQ ID NO.：6
		PBGD encoding sequence 7	SEQ ID NO.：7
PBGD encoding sequence 8	SEQ ID NO.：8
		PBGD encoding sequence 9	SEQ ID NO.：9
PBGD encoding sequence 10	SEQ ID NO.：10
		The PBGD encoding sequence, GenBank accession number X04217	SEQ ID NO.：11
The PBGD encoding sequence, GenBank accession number X04808	SEQ ID NO.：12
		The PBGD encoding sequence, GenBank accession number M95623	SEQ ID NO.：13
PBGD is from the aminoacid sequence of encoding sequence, GenBank accession number M95623, composing type form	SEQ ID NO.：14
		PBGD is from the aminoacid sequence of encoding sequence, GenBank accession number M95623, erythropoiesis form	SEQ ID NO.：15
ASA encoding sequence GenBank accession number J04593	SEQ ID NO.：16
		The SEQ ID NO.:1 of ASA encoding sequence WO 2005/073367	SEQ ID NO.：17
The SEQ ID NO.:2 of ASA aminoacid sequence WO 2005/073367	SEQ ID NO.：18
		The SEQ ID NO.:3 of ASA aminoacid sequence WO 2005/073367	SEQ ID NO.：19
The SEQ ID NO.:4 of ASA aminoacid sequence WO 2005/073367	SEQ ID NO.：20
		The SEQ ID NO.:1 of LAMAN aminoacid sequence WO 2005/094874	SEQ ID NO.：21
The SEQ ID NO.:1 of LAMAN encoding sequence WO 2005/094874	SEQ ID NO.：22
		The galactocerebrosidase encoding sequence	SEQ ID NO.：23
The galactocerebrosidase aminoacid sequence	SEQ ID NO.：24

With reference to these sequences, polypeptide of interest comprises the amino acid that is selected from the following group of forming according to the preferred embodiment of the present invention:

I) as SEQ ID NO.:14, SEQ ID NO.:15, SEQ ID NO.:18, SEQ ID NO.:19, SEQ ID NO.:20, SEQ ID NO.:21 and the defined arbitrary amino acid sequence of SEQ ID NO.:24;

Ii) as i) in the function equivalent part of definition aminoacid sequence; And

Iii) as i) or ii) in the function equivalent analogue of institute's definition aminoacid sequence, the aminoacid sequence of described analogue with as i) or ii) middle definition aminoacid sequence at least 75% identical.

In embodiment, analogue in iii) with as i) or ii) in the definition sequence at least 80% identical, for example at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or for example at least 99.5% with as i) or ii) in the sequence of definition identical.

In addition, polypeptide of interest can be used and comprise the nucleotide sequence that is selected from following sequence and obtain by recombinant expressed:

I) as SEQ ID NO.:1-13, SEQ ID NO.:16, SEQ ID NO.:17, SEQ ID NO.:22 and the defined any nucleotide sequence of SEQ ID NO.:23;

Ii) with as i) in the identical nucleotide sequence of definition nucleotide sequence at least 75%.

Be that reorganization produces described polypeptide, also preferably the nucleotide sequence in ii) with as i) or the ii) middle sequence at least 80% that defines identical, as at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or as at least 99.5% with as i) or the ii) middle sequence that defines identical.

The composition that comprises polypeptide of interest

Below the description of the composition that comprises polypeptide of interest related to comprise according to the inventive method composition of spissated polypeptide in addition, and this description also relates to and comprises the present composition of 10mg/ml polypeptide of interest at least.

The present invention also relates to comprise the composition of 10mg/ml polypeptide of interest at least, wherein, polypeptide of interest can be according to any polypeptide of the present invention, especially as rhPBGD, aryl sulphatase, alpha-Mannosidase or galactocerebrosidase.Said composition especially can comprise 25mg/ml polypeptide of interest at least, as 50mg/ml or 75mg/ml or 100mg/ml polypeptide of interest at least at least at least.Therefore, said composition especially can be included in the polypeptide of interest between the 10-1000mg/ml, as between the 10-500mg/ml or between the 10-300mg/ml or between the 10-200mg/ml or between the 25-500mg/ml or between the 25-400mg/ml or between the 40-400mg/ml or between the 40-300mg/ml or between the 50-400mg/ml or between the 50-300mg/ml or between the 75-400mg/ml or between the 75-300mg/ml or in the polypeptide of interest between the 100-200mg/ml or between 100-150mg/ml.

The composition that comprises polypeptide of interest especially can be the aqueous solution.

Except the polypeptide that comprises high density, said composition especially can also not comprise the coacervate of polypeptide of interest or at least only few coacervate.Therefore, the amount of the polypeptide of interest that exists as coacervate especially can account for polypeptide of interest total amount in the composition less than 5w/w%.Especially described coacervate can account for less than 4w/w%, as less than 3w/w% or less than 2w/w% or less than 1w/w% or less than 0.5w/w% or less than the polypeptide of interest total amount of 0.1w/w%.In this paper environment, term " coacervate " means any form of the polypeptide of interest of non-monosomy.Therefore, this term term comprises any dimer or the polymer of polypeptide of interest.

In addition, if described composition only comprises polypeptide of interest or other protein of trace (promptly differing from the protein of polypeptide of interest) at least only, then favourable.Therefore in concrete mode, said composition comprises except that polypeptide of interest less than 1w/w%, as less than 0.5w/w% or less than 0.1w/w% or less than 0.05w/w% or less than other protein of 0.01w/w%.

Series of factors influences the stable and active of polypeptide, and comprises the composition of polypeptide of interest so can especially be optimized to keep polypeptide of interest as far as possible stable.

PH influences the stability of polypeptide of interest usually, therefore the pH that comprises the composition of polypeptide of interest especially can be in the 7.5-8.5 scope, as specifically between pH 7.7-8.2, more specifically between the pH 7.8-8.0 or between pH 7.85-7.95, as pH 7.8 or pH 7.9.If polypeptide of interest is PBGD, pH especially can be such.

Therefore, the composition that comprises polypeptide of interest especially can comprise and can keep the buffer reagent of composition in described pH scope.The example of this type of buffer reagent includes but not limited to three (methylol) aminomethane hydrochloride (TRIS-HCL), Trisodium Citrate (Na-citrate) and Na ₂HPO ₄The concentration of sort buffer agent can depend on the existence to other composition in the selection of concrete buffer reagent and the composition.If buffer reagent is Na ₂HPO ₄, Na ₂HPO ₄Concentration can be 0.5-15mM such as 1-10mM, or 1.5-7.5mM such as 1.83-7.4mM, or 1.5-3mM such as 1.83-3.7mM, or 1.83-2.45mM, or 3.5-7.5mM such as 3.6-7.4mM, or 5.4-7.4mM, as 1.84mM or 2.45mM or 3.67mM or 5.51mM or 7.34mM.

If buffer reagent is TRIS-HCL, it especially can be 2-50mM for the concentration of TRIS-HCL, as 2-40mM or 2-30mM or 2-20mM or 2-10mM or 5-25mM or 5-20mM or 8-12mM or 9-11mM, for example 10mM.

Comprise other examples for compounds that the composition of polypeptide of interest can comprise and include but not limited to amino acid, sugar, pure and mild washing agent.The example of suitable combination thing includes but not limited to glycine, N.F,USP MANNITOL, sucrose, L-Serine, Tween 80 or one or more described combination of compounds.These compound concentrations depend on particular compound, but for glycine, concentration especially can be 1-200mM, as 5-190mM or 10-180mM or 10-170mM or 20-160mM or 20-150mM or 25-125mM, perhaps 5-100mM or 5-90mM or 5-80mM or 5-70mM or 5-60mM, perhaps 10-100mM or 10-90mM or 10-80mM or 10-70mM or 10-60mM or 12-60mM or 12-55mM or 13.5-54mM or 10-30mM, as 13.5-27mM or 13.5-18mM, or 25-55mM such as 27-54mM, or 40-55 such as 40.5-54mM, as 12.5mM, 13mM, 13.5mM, 14mM, 14.5mM, 17mM, 17.5mM, 18mM, 18.5mM, 19mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 39.5mM, 40mM, 40.5mM, 41mM, 41.5mM or 53mM, 53.5mM, 53mM, 54.5mM or 55mM.

The concentration of N.F,USP MANNITOL especially can be 50-1000mM, as 50-900mM or 50-800mM or 50-700mM or 50-600mM or 100-900mM or 100-800mM or 100-700mM or 100-600mM or 100-500mM or 120-525mM or 125-500mM or 100-300mM, as 120-275mM or 120-170mM or 200-600mM, as 225-550mM or 240-510mM or 370-525mM, as 120mM, 125mM, 130mM, 160mM, 165mM, 166.7mM, 170mM, 175mM, 200mM, 221mM, 225mM, 250mM, 275mM, 300mM, 365mM, 370mM, 375mM, 380mM, 385mM, 490mM, 495mM, 500mM, 505mM or 510mM.

Concentration of sucrose especially can be 1-200mM, as 5-190mM or 10-180mM or 10-170mM or 20-160mM or 20-150mM or 25-125mM or 5-100mM or 5-90mM or 5-80mM or 5-70mM or 5-60mM or 10-100mM or 10-90mM or 10-80mM or 10-70mM or 10-60mM or 12-60mM or 12-55mM or 13.5-54mM or 10-30mM, as 13.5-27mM or 13.5-18mM or 25-55mM, as 27-54mM or 40-55, as 40.5-54mM, as 12.5mM, 13mM, 13.5mM, 14mM, 14.5mM, 17mM, 17.5mM, 18mM, 18.5mM, 19mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, 39.5mM, 40mM, 40.5mM, 41mM, 41.5mM or 53mM, 53.5mM, 53mM, 54.5mM or 55mM.If sucrose is included in the middle of the composition that also comprises N.F,USP MANNITOL, then the concentration of N.F,USP MANNITOL especially can reduce corresponding to concentration of sucrose; Be that the mannose concentration of N.F,USP MANNITOL especially can will use separately with N.F,USP MANNITOL with the merging concentration of sucrose the time is identical.

The concentration of Tween 80 especially can be 0.001-1w/v%, as 0.005-1w/v% or 0.01-1w/v% or 0.001-0.5w/v% or 0.005-0.5w/v% or 0.01-0.5w/v% or 0.05-0.4w/v% or 0.05-0.3w/v% or 0.05-0.2w/v% or 0.075-0.4w/v% or 0.075-0.3w/v% or 0.075-0.2w/v% or 0.09-0.2w/v%, as 0.075w/v%, 0.08w/v%, 0.09w/v%, 0.1w/v%, 0.125w/v%, 0.15w/v%, 0.175w/v% or 0.2w/v%.

The composition that comprises polypeptide of interest, wherein polypeptide especially can be PBGD, aryl sulphatase, lysosome alpha-Mannosidase or galactocerebrosidase, especially can comprise one or more above mentioned combination of compounds.So the suitable example of composition can be also to comprise Na except that polypeptide of interest ₂HPO ₄, glycine and N.F,USP MANNITOL a kind of composition.The pH of said composition can be as mentioned above with different compound concentrations.Therefore, described composition can comprise 0.5-15mM Na in one embodiment ₂HPO ₄, 1-200mM glycine, 50-1000mM N.F,USP MANNITOL and have the pH of 7.5-8.5.Any combination of compound concentration referred to above and pH is by the present invention includes.The specific examples of other compound and pH appropriate combination is to comprise 3.67mM Na in comprising the composition of polypeptide of interest ₂HPO ₄, 27mM glycine, 250mM N.F,USP MANNITOL and have a kind of composition of 7.7-7.9pH.

Other example of suitable groups compound includes, but are not limited to following any example:

1.84mM Na ₂HPO ₄, 13.5mM glycine, 125mM N.F,USP MANNITOL and pH7.7-7.9.

2.45mM Na ₂HPO ₄, 18mM glycine, 167mM N.F,USP MANNITOL and pH 7.7-7.9.

5.51mM Na ₂HPO ₄, 40.5mM glycine, 375mM N.F,USP MANNITOL and pH7.7-7.9.

7.34mM Na ₂HPO ₄, 54mM glycine, 500mM N.F,USP MANNITOL and pH 7.7-7.9.

3.67mM Na ₂HPO ₄, 27mM glycine, 220mM N.F,USP MANNITOL, 30mM sucrose and pH 7.7-7.9.

3.67mM Na ₂HPO ₄, 245mM N.F,USP MANNITOL, 32mM sucrose and pH 7.7-7.9.

3.67mM Na ₂HPO ₄, 27mM L-Serine, 250mM N.F,USP MANNITOL and pH7.7-7.9.

10mM TRIS-HCl, 27mM glycine, 250mM N.F,USP MANNITOL and pH 7.7-7.9.

3.67mM NaCitrat, 27mM glycine, 250mM N.F,USP MANNITOL and pH 7.7-7.9.

3.67mM Na ₂HPO ₄, 27mM glycine, 220mM N.F,USP MANNITOL, 29mM sucrose, 0.1% (w/v) Tween 80 and pH 7.7-7.9.

3.67mM Na ₂HPO ₄, 27mM glycine, 220mM N.F,USP MANNITOL, 29mM sucrose, 0.1% (w/v) Tween 80 and pH 7.7-7.9.

The composition that comprises polypeptide of interest especially can be used for the therapeutic application of Mammals.Therefore, the composition that comprises polypeptide of interest can be isoosmotic with respect to mammiferous tissue especially, for example it especially can have 200-400mOsm/kg, as 250-350mOsm/kg or 275-325mOsm/kg or 295-305mOsm/kg, as the Osmolality of 295mOsm/kg or 300mOsm/kg or 305mOsm/kg.

Concentrate the method for polypeptide of interest

Method of the present invention comprises a) centrifugal and/or filter composition and the b comprise polypeptide of interest) concentrate step from the composition of step a).The present inventor has been found that the composition that comprises polypeptide of interest by centrifugal and/or filtration, concentrate said composition subsequently, might obtain to comprise highly spissated polypeptide of interest and do not have any coacervate of polypeptide of interest or the composition of a small amount of coacervate is only arranged.In addition, use for the therapeutic of polypeptide usually and advantageously reduce the polypeptide coacervate, for example because they can increase the risk that excites at the immunne response of described polypeptide.

Be the subcutaneous administration polypeptide, advantageously peptide composition has high reactivity in small volume, because only small volume can be done subcutaneous injection.

Protein or polypeptide can form coacervate usually when concentrated.Therefore advantageously when using method of the present invention to concentrate polypeptide of interest, described method does not cause the polypeptide coacervate formation of height ratio.As shown in embodiment, similar to the amount of the PBGD coacervate of unconcentrated PBGD composition by the amount of the PBGD coacervate in the composition of concentration method acquisition of the present invention.

In concrete mode, the step a) of described method is carried out prior to step b).

Centrifugal and/or the filtration of step a)

The present inventor found concentrate comprise the composition of polypeptide of interest before, by centrifugal and/or filtration and this composition of pre-treatment is favourable,, removed numerous or most polypeptide coacervates because by this pre-treatment.

When the composition in the step b) concentrates when depending on the method (as ultrafiltration process) of using filter or film and carry out,, the existence of coacervate can not pass through filter or film to such an extent as to can stopping up filter or film small molecules and liquid.This may reduce the spissated speed of composition and/or thoroughly stop any further concentrated.

Therefore, concentrating for such, is favourable according to the pre-treatment of step a), might obtain such polypeptide of interest composition because remove coacervate, more concentrated when it does not do pre-treatment than composition.

When the composition in the step b) concentrates by based on removing the method (as lyophilization or method of evaporation) of anhydrating when carrying out, the pre-treatment in the step a) is favourable, is that pre-treatment has reduced the amount of the coacervate that exists in the concentrate composition.

Step a) can be undertaken by one of following three kinds of methods:

It is centrifugal,

Filter, or

Centrifugal and filter.

Comprise centrifugal and filter as step a), advantageously centrifugally before filtering, carry out because the present inventor found centrifugal removed most large volume coacervates and filter removed subsequently still exist than small agglomerates.

Centrifugal

For removing coacervate, the composition that comprises polypeptide of interest can be in the power scope of 1500-3000g, and is centrifugal as carrying out in the power scope of 1800-2500g or 2000-2300g.

Generally, can be with the centrifugal 10-60 of composition minute, as 15-50 minute or 20-40 minute.

Because temperature can influence the stability of polypeptide of interest, centrifugal can carrying out on 3-15 ℃ or 3-10 ℃ or 3-8 ℃ at temperature 2-20 ℃ is as carrying out at 4 ℃ or 5 ℃ or 6 ℃.

The centrifugal polypeptide of interest coacervate precipitation that causes, promptly they form throw out, and one polypeptide of interest molecule is stayed in the solution.Therefore the supernatant liquor of centrifugal composition uses subsequently in the methods of the invention just.

Filter

The composition that comprises polypeptide of interest can be through having a 0.20-5 μ m aperture, filters as the filter in 0.2-2.5 μ m aperture.

Except that the aperture of filter, the material of manufacturing filter may influence the filtration to polypeptide of interest.The example of suitable membrane filter includes but not limited to polyethersulfone (PES), rhodia, reproducibility Mierocrystalline cellulose and poly(vinylidene fluoride) (PVDF).

When molecule such as protein are filtered, remove small molecules usually, therefore after filtration, polypeptide of interest may reside in the retentate usually.Thereby in subsequent step of the present invention, use usually from filtering retentate.

Step b) concentrates

In principle, any means that concentrates the polypeptide of interest composition can be used in step b) of the present invention.

The example of this type of appropriate method includes but not limited to ultrafiltration process and by removing the method for enrichment anhydrate.

Ultrafiltration

Ultrafiltration is wherein to use hydraulic pressure to pass the separation method of film to force molecule and solvent, and wherein said film comprises the specific size hole of (be called again and hold back sizes values (cut-off value)).The molecule that only has the molecular weight littler than the cutoff value of film can pass this film, and those molecules that molecular weight is bigger are only then worn this film and formed so-called retentate.Thereby the molecule that is present in the retentate is concentrated because solvent streams crosses film.

In embodiment, can pass through tangential flow filtration method (TFF) to the solution or the concentrating of composition that comprise polypeptide of interest.This method is particularly useful for extensive concentrating, and promptly is used for concentrating having 1 liter-hundreds of solution that rise volume.Therefore this method is particularly useful in commercial quantity producing the concentrated solution of polypeptide of interest.

The TFF technology makes solution stream to be filtered cross the special device semi-permeable membranes of semi-permeable membranes based on utilization; Only little than fenestra molecule can pass through film, forms filtrate, stays big material (retentate) to be collected.For the TFF method, apply two kinds of different pressure; A kind of pressure pumps into system with solution and makes it in system circulation (inlet pressure), and another kind of pressure is applied on the film (film pressure) and passes film to force molecule and solvent.Inlet pressure can be 1-3bar generally, as 1.5-2bar.Film pressure generally can be greater than 1bar.

When TFF was used for concentrating said composition, the concentrate composition of polypeptide of interest can be collected as retentate.

The film that is used for TFF can be made by reproducibility Mierocrystalline cellulose or polyethersulfone (PES) usually.

The aperture of film generally can have less than 10.000Mw, as the molecular weight cutoff value of 10-10.000Mw.

In another embodiment, can be undertaken by using centrifugal device the concentrating of composition that comprises polypeptide of interest.The principle of this method is that solution filters because of film is applied centrifugal force on film.This type of film often cuts value by molecular weight (Mw) and characterizes, and promptly this molecular weight cutoff value is can pass the out to out of compound of film and molecular size will can not pass this film greater than the compound of this value.The Mw cutoff value of used film especially can be less than 30000Mw, as between 10-30000Mw among the present invention.

Described film especially can be made by polyethersulfone (PES) or reproducibility Mierocrystalline cellulose.

So suitable commercial filtration unit example can be Centricon Plus-80 or CentriconPlus-15.

Concentrating generally can be by at 2000-4500g, as 2500-4000g or 2750-3500g or 3000-3500g, as centrifugal on 3000g or 3100g or 3200g or 3300g or 3400g or 3500g and carry out.

Generally, centrifugal can carrying out several hours for example continues to surpass 1 hour, as continuing 1-10 hour.

In order to make any disadvantageous effect minimize, centrifugally specifically can carry out on as 3-15 ℃ or 3-10 ℃ or 3-6 ℃ at temperature 2-20 ℃ to polypeptide of interest stability.

It is concentrated to anhydrate

The spissated principle of anhydrating is normally removed whole or most water obtaining solid, and subsequently with this solid dilution or be dissolved in the water of volume like this, and wherein said volume of water is less than this solid before dilution or dissolved volume of water therein.But in principle, the water that this can be by only removing necessary amount need not subsequently redilution or dissolves described compound again to carry out to obtain desired concn.

The example of the spissated appropriate method of anhydrating comprises lyophilization and method of evaporation.

Three most important parameters for lyophilization and method of evaporation are temperature, pressure and time.

Freeze drying process can comprise following three or four steps: freezing stage, elementary drying stage and secondary drying stage and choose annealing steps after the freezing stage wantonly.Freeze-drying especially can be as other step of the inventive method and as described in the lyophilization that comprises and carry out.

Other step

Polypeptide of interest can derive from natural origin, promptly especially can be recombinant expressed from the cell or the polypeptide of interest of natural expression polypeptide of interest.

No matter wherefrom, polypeptide of interest can be subjected to purifying before handling accepting the inventive method.

This " purifying " especially can include but not limited to remove the cell relic, remove do not comprise polypeptide of interest in assorted other protein and remove other composition that can in polypeptide of interest derives from wherein source, exist.Therefore in the specific embodiment of the present invention, the composition that comprises polypeptide of interest comprises less than 5w/w% or less than 1w/w% or less 0.5w/w% or less than 0.1w/w% or less than 0.05w/w% or less than other protein except that polypeptide of interest of 0.01w/w%.

Therefore, can from the composition that comprises polypeptide of interest, remove other protein, in the bright method of we, use them subsequently by for example host cell expression.

Therefore in embodiment, method of the present invention can be included in the one or more the following steps before the step a):

I) recombinant expressed polypeptide of interest,

Ii) by one or more chromatographic step purifying polypeptide of interest compositions,

Iii) change preparation damping fluid (formulation buffer).

Recombinant expressed polypeptide of interest especially can be as before to as described in the polypeptide of interest and carry out.

If polypeptide of interest is PBGD, the chromatography example of adequate types includes but not limited to affinity chromatography, ion exchange chromatography (IEC) and the chromatography on hydroxyapatite column.Can use the arbitrary combination of these chromatography methods in principle.Advantageously carry out affinity chromatography step and this step and any other chromatography method combination at least if the present inventor has before had been found that for PBGD, then advantageously before other chromatographic step, carried out affinity chromatography step (seeing for example WO 03/002731).

For polypeptide of interest wherein is the embodiment of PBGD, and the example of commercially available affinity column comprises affinity coupling post, group specificity affinity column and metallo-chelate affinity column.

Amoxicillin Pharmacia biotechnology (Amersham Pharmacia Biotech) company's calendar year 2001 products catalogue provides the example of affinity coupling post, as comprises band-NH ₂Fixed ligand post and comprise the post of the part of being with primary amine groups.

The metallo-chelate affinity column is particularly preferred for by forming metal ion complex and protein purification with the Histidine group that exposes.The embodiment 3 of WO01/07065 has described the recombinant human porphobilinogen deaminase that makes up band " His-Tag ".For purifying rhPBGD-His, preferably use the metallo-chelate affinity column, as have the post of the affine resin of cobalt metal.

The example of the affinity chromatography method that other is suitable includes but not limited to the pork liver element as the post of part or with 1-amino-4-[[4-[[4-chloro-6-[[3 (or 4)-thio-phenyl] amino]-1,3,5-triazine-2-yl] amino]-the 3-thio-phenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracene sulfonic acid (being called Cibracon Blue 3G again) is as part and use the post of triazine coupling method as the ligand coupling method.The commercial examples of a kind of post in back is that the blue-sepharose gel 6 from Amersham Pharmacia Biotech flows (Blue Sepharose 6 FastFlow) (FF) fast.Therefore, the preferred embodiment of the present invention relates to method as described herein, and wherein the affinity column of step (i) is to use the post of triazine coupling method as the ligand coupling method, and more preferably wherein part be Cibacron Blue 3G.

Term " ion exchange chromatography (IEC) " should be interpreted as such post according to this area in this article, and it is based on the net charge of this post on certain pH, the static by the charged group on the post therewith in conjunction with and isolated molecule such as protein.Ion-exchange refers to one type ionic adsorption is lost other ion to the solution with exchange to the post.

The example of suitable IEC post is such post, as Q Sepharose post, Q SP Sepharose post or CM Sepharose post, especially can be diethylamino agarose (DEAE Sepharose) post.

The example of suitable hydroxyapatite column is ceramic hydroxyapatite column.Hydroxyapatite (Ca ₅(PO ₄) 3OH) ₂Be to be used for separation and protein purification, enzyme, nucleic acid, virus and other macromolecular a kind of calcium phosphate form.The pottery hydroxyapatite is spherical, the big well format of hydroxyapatite.CHT I type (Bole (Bio-Rad)) is the example of suitable commercially available ceramic hydroxyapatite chromatography post.

In one embodiment, method of the present invention can be included in step a) the following step before:

I) recombinant expressed PBGD;

Ii) make from step I) the PBGD composition accept affinity chromatography;

Iii) make step I i) the PBGD composition accept ion exchange chromatography;

In another embodiment, method of the present invention can be included in the following step before the step a):

I) recombinant expressed PBGD;

Ii) make from step I) the PBGD composition accept affinity chromatography;

Iii) make from step I i) the PBGD composition accept ion exchange chromatography;

Iv) make from step I PBGD composition ii) and accept hydroxyapatite column.

These two kinds of methods can comprise randomly that dilution or diafiltration are from step I i) other step of the PBGD composition that obtains.Therefore described step should be at step I i) afterwards and before iii), i.e. step I ia.Step I ia) purpose is to make salt concn be reduced to suitable specific conductivity, for example＜and 10mS/cm.When using DEAE Sepharose as the ion exchange chromatography step to be the resin of step I in ii), this may be especially important, because this can promote the PBGD that the caught combination to DEAE Sepharose resin.Dilution can obtain by direct interpolation purified water or by carrying out ultrafiltration at purified water.

Step I) recombinant expressed PBGD can be undertaken by any means mentioned above.

At step I i) in the example of suitable affinity column can be any affinity column as mentioned above.

The example of carrying out the appropriate method of ion exchange chromatography in step I in ii) can be an any means as mentioned above.

Example at the suitable hydroxyapatite chromatography post of step I in v) can be any hydroxyapatite chromatography post as mentioned above.

In embodiment, affinity column can be such post, and it uses the triazine coupling method as the ligand coupling method, and especially wherein part is the method for Cibacron Blue 3G.This post especially can be Blue Sepharose 6 Fast Flow posts, and ion exchange column can be the DEAESepharose post, and described therein method comprises also in the step I embodiment v) that this post especially can be ceramic hydroxyapatite column.

Method of the present invention also can be included in step b) other step afterwards of this method.This type of step includes but not limited to one or more the following steps:

Freeze-drying comprises the composition that concentrates polypeptide of interest,

Change the buffer reagent that comprises the composition that concentrates polypeptide of interest,

Sterile filtration comprises the composition that concentrates polypeptide of interest,

Evaporation.

Different Freeze Drying Equipments be can use in the methods of the invention, volume and other parameter of freeze-drying solution treated.The example of suitable Freeze Drying Equipment includes but not limited to be used as Lyostar (FTM-Systems) Freeze Drying Equipment of example of the present invention, and the solution that wherein will comprise concentrated polypeptide of interest (being PBGD promptly) in this example is filled in 2ml and the 6ml injection vial (1 type) and with rubber (chloroprene rubber) plug to be sealed.Freeze-drying can be undertaken by following three steps;

It is i) freezing,

Ii) elementary drying, and

Iii) secondary drying.

Step I) freezingly especially can be undertaken by mode like this, i.e. after this load sample and sample is cooled to 0 ℃ and kept 30 minutes at 0 ℃ at first reduces temperature to-40 ℃ and sample was kept 30 minutes at-40 ℃ with 1 ℃ of per minute.

Step I i) elementary drying especially can be undertaken by mode like this, promptly is pumped into the 126mTorr vacuum pressure, keeps 360 minutes at 0 ℃ with 1 ℃ of elevated temperature to 0 of per minute ℃ and with sample.

The ii) secondary drying of step I especially can be undertaken by mode like this, promptly is pumped into the perfect vacuum, simultaneously with 0.5 ℃ of elevated temperature of per minute to+30 ℃ and sample kept 360 minutes at+30 ℃.

After secondary drying, sample can further seal at the vacuum lower seal or after filling with nitrogen.

The example of suitable freeze drying process is included in a kind of method of describing in the example of the present invention.

Freeze-drying can be included in the annealing steps before the elementary dry phase in other embodiments.The present inventor has found in freeze drying process to improve visual appearance comprising of annealing steps, as sees crackle still less, and/or compares during with the identical freeze drying process that uses no annealing steps, causes the reconstitution time of lyophilized products shorter.The time that shortens the reconstruct lyophilized products is favourable, especially when drug use that the lyophilized products conduct is used in the solution mode.For most products, the visual appearance of improvement is regarded advantage usually as.

Therefore, freeze-drying can comprise the following steps:

It is i) freezing,

Ii) annealing,

Iii) elementary drying,

Iv) secondary drying.

Freezing, elementary drying and secondary drying step especially can carry out as mentioned above.Annealing steps is step I i) especially can be undertaken by mode like this, promptly after freezing 30 minutes, with the speed elevated temperature of 2 ℃ of per minutes for example to-10 ℃ or-20 ℃ and keep this temperature to continue 120 minutes or 420 minutes, and the speed with 2 ℃ of per minutes for example reduces temperature to-40 ℃ subsequently, on this temperature, sample can be kept 60-90 minute, after this begin elementary drying step.

Changing that the buffer reagent comprise the composition that concentrates polypeptide of interest especially can undertake by mode like this can, be a) will comprise the composition that concentrates polypeptide of interest in buffer reagent or preparation, to dilute for example 5-15 times, b) once more the composition of concentration and dilution and steps performed a) and b) enough number of times, to such an extent as to the amount that was present in the vehicle in buffer reagent or preparation in the composition before these steps is less than for example 5v/v% or be present in the vehicle in buffer reagent or preparation in the composition less than 1v/v% after described step is carried out.

Especially comprise from the present composition of polypeptide of interest that step b) obtains and also specifically to comprise the step of the described composition of sterile filtration and/or the step of freeze-drying said composition.

Sterile filtration is undertaken by composition is filtered through the filter with 0.22 μ m or 0.20 μ m aperture usually.Freeze-drying can be carried out especially as mentioned above.

The present invention also relates to lyophilised compositions by the inventive method acquisition.

Subcutaneous injection

The composition that the present invention also relates to comprise the 50-300mg/ml polypeptide of interest is used to make in order to the purposes of subcutaneous injection to mammiferous medicine.

Polypeptide of interest can be any polypeptide of interest of the present invention, and it includes but not limited to PBGD, aryl sulphatase A, lysosome alpha-Mannosidase and galactocerebrosidase.

Term is subcutaneous often writes a Chinese character in simplified form into s.c. and this two terms can exchange use in the context of the present invention.

When injection is implemented in subcutaneous mode, can not injection surpass 1.5mL because of the physiological restriction usually.

Because the patient usually needs a certain amount of specific polypeptide of interest, existence is related between the peptide concentration of purpose in the volume of the composition that comprises the polypeptide of interest that need be applied to the patient and described composition.

Therefore the composition that the present invention is advantageously contained polypeptide of interest comprises the polypeptide of interest of high density and the polypeptide of interest of this high density can obtain under the situation that does not form a large amount of polypeptide coacervates.The use of this type of concentrated polypeptide of interest composition might realize following situation, promptly injects the described composition of small volume more and guarantees that meanwhile the patient accepts the polypeptide of interest of capacity; So easier subcutaneous administration polypeptide of interest.

The above-mentioned composition that comprises polypeptide of interest especially can comprise 75-250mg/ml, as the polypeptide of interest of 75-200mg/ml or 75-150mg/ml or 100-150mg/ml or 100-125mg/ml or 125-150mg/ml.

As mentioned above, the volume of composition that comprises polypeptide of interest is relevant with the concentration of polypeptide of interest in the described composition, wherein needs to inject described volume to accepting the capacity polypeptide of interest to the patient to guarantee this patient.

Thereby the volume of this composition will be regulated according to the concentration of polypeptide of interest in the composition usually.Yet described volume can be 0.1-1.5ml usually, as 0.1-1.5ml or 0.5-1.5ml or 0.5-1.5ml or 0.75-1.5ml or 0.75-1.5ml or 1-1.5ml or 1-1.5ml.

The amount of polypeptide of interest is important for being applied to the patient, and it depends on whose body weight and specific polypeptide of interest usually.

In one embodiment, the present invention relates to treat the method for Mammals acute intermittent porphyria, it comprises that subcutaneous injection contains the composition of 50-300mg/ml PBGD.

Using especially of PBGD can be used for the treatment of acute intermittent porphyria.Yet using also of PBGD of design can be used for the treatment of other porphyria, as hereditary coproporphyrin or variegate porphyria.Porphyria is the term that is used for being referred to as by different multiple diseases due to damaged in the protoheme biosynthesizing way.What therefore, can conceive is to use the integral body conversion that PBGD (for example with other therapeutical agent combination) can help to increase different intermediates in the protoheme biosynthesizing way to the patient who suffers from any kind porphyria.Meissner PN etc. for example, 1986, clinical study Europe magazine (European Journal of Clinical Investigation), the 16th volume, 257-261; Hift RJ etc., 1997, S.Afr.Med.J., the 87th volume, 718-27 and MeissnerP etc., 1993, Journal of Clinical Investigation (J.Clin.Invest.), the 91st volume, 1436-44 has described ALA and the accumulation of PBG in hereditary coproporphyrin and variegate porphyria.In these diseases, the accumulation of ALA and PBG is respectively because of forming due to the enzyme defect that is positioned at downstream the 4th and the 5th step that ALA to PBG transforms.In two pieces of up-to-date papers, described the porphyrinogen that in the variegate porphyria patient, accumulates and how can suppress PBG-Deaminase.

In another embodiment, the present invention relates to treat the method for Mammals metachromatic leukodystrophy, it comprises that subcutaneous injection contains the composition of 50-300mg/ml aryl sulphatase A.

Metachromatic leukodystrophy (MLD) is caused by the autosomal recessive inheritance defective among the lysosomal enzyme aryl sulphatase A (ASA), causes myelin film carrying out property destruction (demyelination) and semi-lactosi glycosyl thioester (galatosyl sulphatide) (cerebroside sulfate) to accumulate in the white matter of central nervous system (CNS) and peripheral nervous system.In the histology prepared product, semi-lactosi glycosyl thioester forms the spherical particle material of metachromatic staining.Semi-lactosi glycosyl thioester also is accumulated in kidney, bladder and some other internal organs and to cross volume to be secreted in urine.

The galactosyl sulfatide is normally by through lysosomal enzyme aryl sulphatase A (EC 3.1.6.8) (journal of biological chemistry (Biochem J) .1964 such as Austin, 93,15C-17C) and be called the combined action of sphingolipid activator of saponification protein B and the in addition metabolism of hydrolysis 3-O-sulfuric acid ester bond to form galactocerebroside.There is (seeing below) in the famine of aryl sulphatase A in the whole tissues from the patient who suffers from baby late period, teenager and adult's form MLD.Hereinafter, aryl sulphatase A albumen will be called " ASA ".The famine of ASA is present in whole tissues from MLD patient.

In another embodiment, the present invention relates to treat the method for Mammals lysosomal storage disease α-mannosidosis, it comprises that subcutaneous injection contains the composition of 50-300mg/ml lysosome alpha-Mannosidase.

α-mannosidosis is with frequency 1/1000000 and the 1/500000 recessive euchromosome disease that takes place at world wide.Mannosidosis is present in whole race's groupings in Europe, America, Africa and Asia.In the whole countries that lysosomal storage disease had good diagnosis service, arrive mannosidosis with similar frequency detecting.They innately are outward appearance health; Yet the symptom of this disease is progressive.α-mannosidosis shows clinical heterogeneity, from the to the utmost point gentle form of utmost point severe form.Typical clinical symptom is: mental retardation, skeleton change, compromised immune (causing repeated infection), hearing loss, and should disease often relevant with typical facial characteristics,, forehead coarse as face protrudes, the bridge of the nose is flat, spur ` and wealthy mouthful.In the serious case of the overwhelming majority (I type mannosidosis), children suffer from hepatosplenomegaly disease and it is dead during 1 year of life.This early stage death may be caused by the severe infections that immunodeficiency causes, and wherein said immunodeficiency is because of due to the mannosidosis.In relatively mild case (2 type mannosidosis), the patient becomes of age usually.Patient's bone weakness causes at 20-40 needs wheelchair during the age in year.This disease causes brain diffusing capacity obstacle, and this often causes the thin and weak [(mental perfomance) of having nothing at all except that the most basic simple reading and writing technical ability.Follow these problems of hearing anergy and other clinical manifestation that the patient can not be lived on one's own life, the result needs long-term care.

In another embodiment, the present invention relates to treat the method for Mammals Krabbe disease, it comprises that subcutaneous injection contains the composition of 50-300mg/ml galactocerebrosidase.

In the people, the shortage of GALC enzyme causes being called the heredity lysosomal storage disease of the autosomal inheritance of Krabbe disease or globoid cell leukodystrophy.This enzyme is expressed in people's testis, kidney, liver and brain usually and the shortage of GALC enzyme causes illness in myelin metabolism and central nervous system and the peripheral nervous system (being respectively CNS and PNS) usually.

In the people of any age, nationality and sex, observe Krabbe disease.

Should be understood that described embodiment and feature also are applicable to others of the present invention under the environment one of aspect the present invention.Especially, describe whole embodiments of the composition that comprises polypeptide of interest, existence, buffer reagent and pH as other compound also are applicable to the composition that comprises used polypeptide of interest among the application.

When one of object of the present invention or its feature or characteristic were mentioned with singulative, this also referred to the plural form of this object or its feature or characteristic.For example, when mentioning " peptide species ", should be understood to refer to one or more polypeptide.

At this specification sheets in the whole text in the scope, vocabulary " comprises " or is out of shape as " comprising " or " comprising " will be understood as hint and comprise described key element, overall or step, or key element, overall or step in groups, but do not get rid of any other key element, overall or step, or key element, overall or step in groups.

The whole patents mentioned in this application and non-references thus in this article by with reference to and intactly incorporate into.

The present invention will describe in following non-limitative experiment part now in more detail.

Experiment

Material

rhPBGD

Obtain the rhPBGD that uses according to the method 2 among the embodiment 1 of WO 03/002731 in following experiment, wherein method 2 is to comprise that step IV is the method for ceramic hydroxyapatite chromatography step.

The preparation damping fluid

The rhPBGD of reorganization and purifying is present in the following aqueous compositions damping fluid:

3.67mM Na ₂HPO ₄

The 27mM glycine

250mM N.F,USP MANNITOL

With pH 7.9

The said preparation damping fluid is with after 0.22 μ m filter carries out sterile filtration.

Method

Freeze-drying

The freeze-drying of purifying rhPBGD solution is carried out according to following scheme in Lyostar (FTM-Systems) Freeze Drying Equipment:

Visual observation (transparency and color)

Liquid carries out visualization study with regard to color, transparency and settling aspect according to following scheme.

Color: 1: colourless; 2: faint yellow; 3: yellow

Transparency: 1: limpid; 2: slightly muddy; 3: muddiness

Other sign: comprise other observations (for example settling, insoluble substance etc.) in some instances here from the operator.

PH-measures

3 kinds of reference solution (Merck) that pH meter (Metrohm 691pH meter) and electrode (LL pH combined electrode) are used in the 4.00-9.00 scope are proofreaied and correct.The described liquid of final analysis.

Protein concn

Protein concn in extract, technological process sample, bulk drug and the end product by as this method mensuration, wherein said method utilized protein in alkaline medium with Cu ²⁺Be reduced into Cu ⁺Principle (Biuret reaction).Cu ⁺Ion reacts with the reductive agent that contains the dihomocinchonine acid (bicinchoninic acid) that causes extremely sensitive and high selectivity colorimetric detection subsequently.

Purity

The percentage composition that recombinant human porphobilinogen deaminase (rhPBGD) and rhPBGD variant depend on organic instrumentality (acetonitrile) in the moving phase according to them adsorbs with the ability of desorption silicon dioxide base mounting medium and separates.

The rhPBGD activity

Porphobilinogen deaminase (PBGD) catalysis is added the porphobilinogen (PBG) of 4 molecules to form the line style tetramer-preceding uroporphyrinogen, and wherein preceding uroporphyrinogen discharges from enzyme and is cyclized into uroporphyrinogen III by the effect of uroporphyrinogen III synthase in vivo.Preceding uroporphyrinogen can carry out chemical oxidation is absorbed in 405nm place light with formation uroporphyrin with benzoquinones.

In each experimental example, a single phial is carried out analysis.Be to measure rhPBGD activity and protein concn, duplicate respectively and in triplicate each phial is tested.

Osmolality

The freeze-drying rhPBGD of a resuspended phial in 1.00ml MilliQ-water.The phial that will contain the refrigerated water solution of rhPBGD melts.3 kinds of standardized solution that osmometer (Vapro osmometer) is used in 100-1000mOsm/kg (100mOsm/kg, 290mOsm/kg, the 1000mOsm/kg) scope are proofreaied and correct.The described liquid of subsequent analysis.

Embodiment 1

Concentrate with centrifugal filter device

Refrigerated PBGD-bulk solution (7mg/mL rhPBGD, 3.67mM Na ₂HPO ₄, the 27mM glycine, 250mM N.F,USP MANNITOL, pH 7.9) in water-bath in 20 ℃ of thawings, under 3200g centrifugal 10 minutes and subsequently by 0.20 μ m-PES filter (Nalgene polyethersulfone filter) sterile filtration.The PBGD-bulk solution is continued several hrs by operation centrifugal filter device Centricon Plus-80 (Mw cutoff value 30000) and Centricon Plus-15 (Mw cutoff value 30000) be concentrated into 100mg/ml on 3200g.Concentrated solution be retentate by 0.22 μ m-filter (Millex GV) sterile filtration and and the most at last the part of this solution dilute to reach 50mg/ml with the sterile preparation damping fluid.The solution of 5mg/ml prepares by the hPBGD that directly dilutes reorganization and purifying with the sterile preparation damping fluid.

Subsequently as mentioned above with the rhPBGD freeze-drying of 5mg/mL, 50mg/mL and 100mg/mL.At 40 ℃ ± 2 ℃, 75% ± 5% relative humidity (RH) down storage contains the several phials of above mentioned freeze-drying rhPBGD solution of 5mg/mL, 50mg/mL and 100mg/mL rhPBGD and the several phials with 5mg/mL rhPBGD aqueous solution separately.Phial lucifuge in second packing material (carton) of fully sealing is stored.

Shown in time point on time point (being period of storage), each freeze-drying sample of a phial is resuspended in 1.00mL Mi Libo (Millipore) water.

With regard to color, transparency and settling aspect each resuspended rhPBGd phial and aqueous rhPBGd phial are made visual observations subsequently, and measure pH as mentioned above, protein concn, purity and rhPBGD activity.

The result provides in following table 1-4:

Table 1: lyophilized products, 5mg/mL

Time point (moon)	Active (U/ml)	Concentration (mg/ML)	Single-minded activity (U/mg)	Purity (%)	Visual observation
						0	93.2	4.3	21.5	99.6	Color: 1, transparency: 1
0.5	81.0	5.2	15.6	ND	Color: 1, transparency: 1
						1	76.6	5.9	13.1	99.9	Color: 1, transparency: 1
1.5	87.0	5.5	15.9	99.7	Color: 1, transparency: 1
						2	53.3	4.7	11.4	99.6	Color: 1, transparency: 1
3	50.8	4.8	10.7	99.6	Color: 1, transparency: 1
						6	34.3	5.3	6.5	99.6	Color: 1, transparency: 1

Table 2: lyophilized products; 50mg/ml

Time point (moon)	Active (U/ml)	Concentration (mg/ML)	Single-minded activity (U/mg)	Purity (%)	Visual observation
						0	888	41.4	21.5	99.1	Color: 2, transparency: 1
0.5	842	50.6	16.6	ND	Color: 2, transparency: 1
						1	746	50,6	14.8	100	Color: 2, transparency: 1
2	640	52.9	12.1	100	Color: 2, transparency: 1
						3	634	49.0	12.9	100	Color: 2, transparency: 1
6	422	43.0	9.8	100	Color: 2, transparency: 1

Table 3: lyophilized products; 100mg/ml

Time point (moon)	Active (U/ml)	Concentration (mg/ML)	Single-minded activity (U/mg)	Purity (%)	Visual observation
						0	1944	83.7	23.2	99.1	Color: 3, transparency: 1
1	1470	98.7	14.9	100	Color: 3, transparency: 1
						2	1282	94.8	13.5	100	Color: 3, transparency: 1
3	1253	82.6	15.2	100	Color: 3, transparency: 1
						6	739	75.5	9.8	100	Color: 3, transparency: 1

Table 4: aqueous product; 5mg/ml

Time point (moon)	Active (U/ml)	Concentration (mg/ML)	Single-minded activity (U/mg)	Purity (%)	Visual observation
						0	95.6	4.0	23.7	99.1	Color: 1, transparency: 1
0.5	48.1	5.4	8.9	ND	Color: 1, transparency: 1
						1	28.6	5.9	4.8	96.1	Color: 1, transparency: 1
1.5	12.3	5.6	2.2	91.4	Color: 1, transparency: 1
						2	4.5	4.4	1.0	90.7	Color: 1, transparency: 1
3	7.1	3.1	2.3	87.3	Color: 2, transparency: 2
						6	4.4	2.1	2.1	58.1	Color: 2, transparency: 2

Embodiment 2

Concentrate the rhPBGD composition by centrifugal filter device

Refrigerated PBGD-bulk solution (7mg/mL rhPBGD, 3.67mM Na ₂HPO ₄, the 27mM glycine, 250mM N.F,USP MANNITOL, pH 7.9) in water-bath in 20 ℃ of thawings, on 3200g centrifugal 10 minutes and subsequently by 0.20 μ m-PES filter (Nalgene polyethersulfone filter) sterile filtration.The PBGD-bulk solution is continued several hrs by operation centrifugal filter device Centricon Plus-80 (Mw cutoff value 30000) and Centricon Plus-15 (Mw cutoff value 30000) be concentrated into 100mg/ml under 3200g.Concentrated solution be retentate by 0.22 μ m-filter (Millex GV) sterile filtration and with preparation damping fluid (seeing above) dilution of sterile filtration to obtain the solution of low concentration.The part that with the volume is every kind of enriched material of unit is carried out freeze-drying as mentioned above.

The different enriched materials of the freeze-drying rhPBGD and the rhPBGD aqueous solution are at 5 ℃ ± 3 ℃ or store at-20 ℃ ± 5 ℃ (ambient relative humidity (RH)).All phial lucifuge in second packing material (carton) of fully sealing is stored.

Shown in time point on time point (being period of storage), be resuspended in each freeze-drying sample of a phial in the 1.00mL Millipore water and observe color, transparency and settling with the aqueous solution of rhPBGD by the range estimation mode subsequently and pH, protein concn, purity, Osmolality and rhPBGD are active to be checked by measuring.

The result provides in following table 5-19:

Table 5: aqueous product; 11mg/ml; Storage temperature :+5 ℃

Table 6: aqueous product: 11mg/ml; Storage temperature :-20 ℃

Table 7: lyophilized products, 11mg/ml; Storage temperature :+5 ℃

Table 8: aqueous product, 17mg/ml; Storage temperature :+5 ℃

Table 9: aqueous product, 17mg/ml; Storage temperature :-20 ℃

Table 10: lyophilized products, 17mg/ml; Storage temperature: 5 ℃

Table 11: aqueous product; 36mg/ml; Storage temperature :+5 ℃

Table 12: aqueous product, 36mg/ml; Storage temperature :-20 ℃

Table 13: lyophilized products, 36mg/ml; Storage temperature: 5 ℃

Table 14: aqueous product, 50mg/ml; Storage temperature: 5 ℃

Table 15: aqueous product, 50mg/ml; Storage temperature :-20 ℃

Table 16: lyophilized products, 50mg/ml; Storage temperature: 5 ℃

Table 17: aqueous product, 100mg/ml; Storage temperature: 5 ℃

Table 18: aqueous product, 100mg/ml; Storage temperature :-20 ℃

Table 19: lyophilized products, 100mg/ml; Storage temperature: 5 ℃

Embodiment 3

Concentrate the rhPBGD composition with tangential flow filtration (TFF)

The bulk solution of rhPBGD is subsequently at 5 ℃ with melted in the dark minimum 3 days.

The solution that melts is used 200mL taper centrifuge tube under 2200g centrifugal about 10 minutes subsequently.

The a series of filters of solution through having following aperture filter: 5.0 μ m; 0.65 μ m; 0.45 μ m and 0.20 μ m concentrate by tangential flow filtration (TFF) subsequently.

TFF concentrates with Mi Libai laboratory scale TFF system (Millipore Labscale TFF System) and Millipore The XL filter exists with pump inlet pressure and the about 4-6psi of about 20-25psi

Pressure on the XL filter and carrying out.RhPBGD is the lucifuge protection by the sampling receptacle that is covered the TFF system by aluminium foil during this method.

The concentrated rhPBGD solution that obtains from the TFF method contains 3.67mM Na at what prepare subsequently in sterilized water ₂HPO ₄.2H ₂The preparation damping fluid of O, 27mM glycine and 220mM N.F,USP MANNITOL carries out buffer reagent and changes.This is by adding described damping fluid continuously to the TFF-system and force it to stride across film to have replaced previous damping fluid until described damping fluid and carry out.

Concentrate the rhPBGD solution that has also changed buffer reagent and do sterile filtration by the filter that makes this solution process have 0.22 μ m aperture subsequently.This sterile filtration carries out twice, uses new filter at every turn.

With aseptic spissated rhPBGD solution with being placed in the phial, after this described in method part and freeze-drying.

Embodiment 4

The amount of different freeze-drying patterns and/or vehicle is to the reconstruct influence of (reconstitution) time

PBGD concentrates and measures the concentration of PBGD as described in example 3 above after changing buffer reagent.

Spissated PBGD solution is freeze-drying in Lyostar (FTM-Systems) Freeze Drying Equipment subsequently.Solution is filled in 2ml and 6ml injection vial (1 type) and seals with rubber plug (chloroprene rubber).

The circulation of freeze-drying originally:

Sample loads at ambient temperature and shelf is cooled to 0 ℃ and continues 30 minutes.Temperature is reduced-40 ℃ (1 ℃ of per minute) and on this temperature, kept 30 minutes, and subsequently vacuum is evacuated to 126mTorr and elementary drying and begins by elevated temperature to 0 ℃ (1 ℃ of a per minute).After elementary dry 360 minutes, temperature is increased to+30 ℃ (0.5 ℃ of per minute) and is pumped into perfect vacuum (beginning secondary drying) simultaneously.Temperature was kept 360 minutes and subsequently phial clogged under vacuum at+30 ℃.

Comprise the freeze-drying of annealing steps:

After continuing 30 minutes on-40 ℃, temperature is reduced to-10 ℃ or-20 ℃ with the speed of 2 ℃ of per minutes, wherein on described temperature, sample was kept 120 minutes or 420 minutes, after this temperature is reduced to-40 ℃ once more with 2 ℃ of per minutes, on this temperature, sample was kept 60-90 minute, after this begin elementary drying.

The result is displayed in Table 20, and is as follows with regard to the circulate meaning of used phrase of vehicle and freeze-drying in table 20:

The vehicle of 1 times of amount refers to that PBGD solution is included in the 3.67mMNa for preparing in the sterilized water ₂HPO ₄* 2H ₂O, 27mM glycine and 220mM N.F,USP MANNITOL.

1.5 doubly Liang vehicle refers to that PBGD solution is included in the 5.51mMNa for preparing in the sterilized water ₂HPO ₄* 2H ₂O, 40.5mM glycine and 375mM N.F,USP MANNITOL, i.e. 1.5 times of every kind of compositions that in 1 * buffer reagent, exist.

2 * vehicle refers to that PBGD solution is included in the 7.34mM Na for preparing in the sterilized water ₂HPO ₄* 2H ₂O, 54mM glycine and 500mM N.F,USP MANNITOL, i.e. 2 times of every kind of compositions that in 1 * buffer reagent, exist.

The circulation of freeze-drying originally as mentioned above.

Annealing freeze-drying circulation as mentioned above, wherein annealing steps comprises and temperature is increased to-10 ℃, sample kept 120 minutes on this temperature, after this temperature was reduced to-40 ℃ again.

The annealing freeze-drying circulation that prolongs as mentioned above, wherein annealing steps comprises and temperature is increased to-20 ℃, sample kept 420 minutes on this temperature, after this temperature was reduced to-40 ℃ again.

Table 20:

Embodiment 5

The amount of different freeze-drying patterns and/or vehicle is to the influence of lyophilized products outward appearance

Concentrate and freeze dried PBGD solution as described in example 4 above and preparation and to the appellation of the amount of vehicle and freeze-drying cyclical patterns have with embodiment 4 in identical meaning.

Following result obtains by the visual inspection lyophilized products:

A: 3 kinds of products that prepare from the solution that comprises 4.6mg/ml 66.6mg/ml and 109.4mg/ml rhPBGD respectively relatively show that the crackle number in the lyophilized products increases with the concentration of rhPBGD.

B: relatively from comprising 150mg/ml rhPBGD and comprising 2 kinds of products that prepare the solution of 1 times of amount and 1.5 times amount vehicle, show crackle number in the lyophilized products for the product that comprises 1.5 times of amount vehicle than the product that comprises 1 times of amount vehicle still less.

C: 2 kinds of products that prepare from the 150mg/ml rhPBGD solution that comprises 1 times of amount and 2 times amount vehicle relatively show that crackle number in the lyophilized products that contains 2 times of amount vehicle is than comprising 1 times of product of measuring vehicle still less.

D: 3 kinds of products for preparing by the annealing freeze-drying circulation that utilizes freeze-drying originally circulation, annealing freeze-drying circulation and prolong from 150mg/ml rhPBGD solution relatively show that crackle number beguine certificate freeze-drying originally in the product that prepare according to annealing freeze-drying circulation in the lyophilized products circulates in the product for preparing still less.In addition, the crackle in the product that prepare according to the annealing freeze-drying circulation that prolongs count beguine according to the annealing freeze-drying circulate in the product for preparing the crackle number still less.

E:3 kind lyophilized products prepares from 150mg/ml, 175mg/ml and 200mg/ml rhPBGD solution respectively.Freeze-drying is carried out with anneal cycles in the vehicle of each self-contained 1.5 times of amount of lyophilized products and they.Lyophilized products does not all comprise any crackle.

F:2 kind freeze-drying rhPBGD product prepares from 150mg/ml rhPBGD solution.Wherein a kind of freeze-drying rhPBGD product comprises the vehicle of 1 times of amount and prepares according to freeze-drying originally circulation, and another kind of freeze-drying rhPBGD product comprises the vehicle of 1.5 times of amounts and prepare according to the annealing freeze-drying circulation that prolongs.The product that comprises the vehicle of 1.5 times of amounts and prepare according to the annealing freeze-drying circulation that prolongs also comprises still less crackle according to the product that the circulation of freeze-drying originally prepares than the vehicle that comprises 1 times of amount.

G:2 kind freeze-drying rhPBGD product prepares from 150mg/ml rhPBGD solution.Wherein a kind of freeze-drying rhPBGD product comprises the vehicle of 1 times of amount and prepares according to freeze-drying originally circulation, and another kind of freeze-drying rhPBGD product comprises with the 0.1%Tween 80 of 1 times of amount excipient composition and according to the annealing freeze-drying circulation that prolongs and prepares.Comprise to comprise than the vehicle that comprises 1 times of amount and according to the product for preparing that circulates of freeze-drying originally and comprise still less crackle with the 0.1%Tween 80 of 1 times of amount excipient composition and according to the product that the annealing freeze-drying circulation that prolongs prepare.

Embodiment 6

The amount of different recovery volumes, vehicle and freeze-drying pattern are to the influence of the stability of freeze-drying rhPBGD

The freeze-drying sample of spissated rhPBGD solution prepares as described in example 4 above.

" bulk solution " is the concentrated solution of PBGD before freeze-drying.

Table 21 shows the result of the rhPBGD solution with following feature: the ratio of the amount of rhPBGD concentration, vehicle (with identical as definition used among the embodiment 4), freeze-drying pattern (with identical as definition used among the embodiment 4) and packing volume (packing volume is the volume of composition before freeze-drying) and recovery volume (recovery volume is that lyophilized products is resuspended in volume wherein).

Solution 1:

About 5mg/ml rhPBGD

The vehicle of 1 times of amount

The circulation of freeze-drying originally

Packing volume=recovery volume

Solution 2:

About 70mg/ml rhPBGD

The vehicle of 1 times of amount

The circulation of freeze-drying originally

Packing volume=2 * recovery volume

Solution 3:

About 110mg/ml rhPBGD

The vehicle of 1 times of amount

The circulation of freeze-drying originally

Packing volume=recovery volume

Solution 4:

About 70mg/ml rhPBGD

The vehicle of 1 times of amount

The circulation of freeze-drying originally

Packing volume=1.5 * recovery volume

Solution 5:

About 90mg/ml rhPBGD

The vehicle of 2/3 times of amount

The circulation of freeze-drying originally

Packing volume=1.5 * recovery volume

Solution 6:

About 60mg/ml rhPBGD

The vehicle of 1/2 times of amount

The circulation of freeze-drying originally

Packing volume=2 * recovery volume

Solution 7:

About 110mg/ml rhPBGD

The vehicle of 1 times of amount

Annealing freeze-drying circulation

Packing volume=recovery volume

Solution 8:

About 60mg/ml rhPBGD

The vehicle of 1 times of amount

Annealing freeze-drying circulation

Packing volume=2 * recovery volume

Solution 9:

About 150mg/ml rhPBGD

The vehicle of 1 times of amount

Annealing freeze-drying circulation

Packing volume=recovery volume

Solution 10:

About 150mg/ml rhPBGD

The vehicle of 1 times of amount

The circulation of freeze-drying originally

Packing volume=recovery volume

Although in table 21, do not show, also each time point has been checked purity, under in the top and bottom, find it is 100%.

Solution 2 has been used wrong recovery volume on the 9th time-of-week point on the 4th week and the 9th time-of-week point and for solution 4.

Table 21:

Solution	Measurement point (week)	Packing volume (ml)	Recovery volume (ml)	pH	Osmolality (mosmol/kg)	Protein concn (mg/ml)	Active (U/ml)	Single-minded activity (U/mg)
									1	Bulk solution					4.6	78	17.1
	0	0.67	0.67	7.54	274	4.8	85	17.8
										2	0.67	0.67	7.22	274	4.6	87	19.4
	4	0.67	0.67	7.78	279	5.1	75	14.5
										7	0.67	0.67	7.87	284	5.1	68	13.3
	9	0.67	0.67	7.67	403	7.0	93	13.2
									2	Bulk solution					66.6	1129	16.9
	0	0.67	0.335	7.64	525	113	1915	16.9
										2	0.67	0.335	7.63	459	93.6	1593	17.0
	4	0.67	0.67	7.75	264	64.6	1104	17.1
										7	0.67	0.335	7.95	451	106.4	2106	19.8
	9	0.67	0.67	7.59	247	51.4	859	16.7
									3	Bulk solution					109.4	1491	13.6
	0	0.67	0.67	7.75	274	99.9	1598	16.0
										2	0.67	0.67	7.64	269	91.4	1543	16.9
	4	0.67	0.67	7.68	274	101.2	1825	18.0
										7	0.67	0.67	7.71	278	103.4	2045	19.8
	9	0.67	0.67	7.67	274	88.3	1656	18.8
									4	Bulk solution					71.5	1244	17.4
	0	0.67	0.45	7.64	448	113.8	1748	15.4
										2	0.67	0.45	7.63	411	86.4	1806	20.9
	4	0.67	0.45	7.77	362	109.9	1897	17.3
										7	0.67	0.45	7.90	379	95.2	686	(7.2)
	9	0.67	0.67	7.63	273	59.7	1090	18.3

5	Bulk solution					91.0	1610	17.7
										0	0.67	0.45	7.65	296	119.4	2014	16.9
	2	0.67	0.45	7.61	285	112.3	2093	18.6
										4	0.67	0.45	7.90	292	125.1	2409	19.3
	7	0.67	0.45	7.88	297	116.4	1928	16.6
										9	0.67	0.45	7.34	278	102.5	1490	14.5
6	Bulk solution					60.7	992	16.3
										0	0.67	0.335	7.63	295	112.6	1753	15.6
	2	0.67	0.335	7.60	288	86.9	1787	20.6
										4	0.67	0.335	7.83	287	116.4	2106	18.1
	7	0.67	0.335	8.20	299	109.7	695	(6.3)
										9	0.67	0.335	7.44	287	95.2	1636	17.2
7	Bulk solution					116.4	1926	16.5
										0	0.67	0.67	7.56	275	101.1	1750	17.3
	2	0.67	0.67	7.51	276	93.4	1831	19.6
										4	0.67	0.67	7.60	270	101.6	1774	17.5
	7	0.67	0.67	7.53	283	102.2	1639	16.0
										9	0.67	0.67	7.46	274	89.9	960	10.7
8	Bulk solution					64.5	1119	17.4
										0	0.67	0.335	7.52	511	100.7	1718	17.1
	2	0.67	0.335	7.51	459	99.3	1900	19.1
										4	0.67	0.335	7.70	482	114.5	1913	16.7
	9	0.67	0.335	7.29	425	102.3	1650	16.1
									9	Bulk solution					165	3587	21.7
	0	0.60	0.60	7.71	309	121.4	2819	23.2
										4	0.60	0.60	7.74		140.3	2014	14.4
	7.5	0.60	0.60	7.61	292	135.9	1640	12.1
									10	Bulk solution					165	3587	21.7
	0	0.60	0.60	7.86	276	142.1	2397	16.9
										3	0.40	0.40	8.20	314	141.9	2381	16.8
	5	0.60	0.60	7.60	302	131.8	2304	17.5

Embodiment 7

Different vehicle is to the influence of rhPBGD stability

RhPBGD concentrate as described in example 4 above and buffer reagent subsequently to one of hereinafter described buffer reagent change.Product is the freeze-drying with included annealing steps originally as described in example 4 above subsequently, and stability of sample is tested as described in example 6 above.

Checked of the influence of following 4 kinds of preparations to rhPBGD stability:

Preparation A (corresponding to the solution among the embodiment 6 9): 250mM N.F,USP MANNITOL, 27mM glycine and 3.67mM Na ₂HPO ₄

Preparation B:250mM N.F,USP MANNITOL, 27mM glycine and 10mM TRIS-HCL.

Formulation C: 250mM N.F,USP MANNITOL, 27mM glycine, 3.67mM Na ₂HPO ₄And 0.1%Tween80.

Preparation D:221mM N.F,USP MANNITOL, 29mM sucrose, 27mM glycine, 3.67mM Na ₂HPO ₄With 0.1%Tween 80.

The result shows in table 22.

Table 22Preparation	Measurement point (week)	Packing volume (ml)	Recovery volume (ml)	pH	Osmolality (mosmol/kg)	Protein concn (mg/ml)	Active (U/ml)	Single-minded activity (U/mg)
									A	Bulk solution			7.69	366	165	3587	21.7
	0	0.60	0.60	7.71	309	121.4	2819	23.2
										4	0.60	0.60	7.74		140.3	2014	14.4
	7.5	0.60	0.60	7.61	292	135.9	1640	12.1
									B	Bulk solution			7.54	320	173	3595	20.8
	0	0.60	0.60	7.58	284	148.1	3726	25.2
										3	0.60	0.60	7.57	280	165.4	2947	17.8
	4	0.60	0.60	7.69		167.5	2367	14.1
										7.5	0.60	0.60	7.60	283	150.4	2235	14.9
C	Bulk solution			7.40	338	178	3606	20.2
										0	0.60	0.60	7.76	290	142.9	2662	18.6
	3	0.60	0.60	7.43	285	181.7	2332	12.8
										4	0.60	0.60	7.42		173.1	1436	8.3
	6	0.60	0.60	7.55	274	156.6	1254	7.4
										7.5	0.60	0.60	7.34	274	141.5	1252	8.9
D	Bulk solution			7.41	337	175	3869	22.1
										0	0.60	0.60	7.80	292	127.5	2355	18.5
	3	0.60	0.60	7.35	288	143.9	1988	13.8
										4	0.60	0.60	7.26		159.3	1644	10.3
	6	0.60	0.60	7.30	281	135.7	1236	9.1
										7.5	0.60	0.60	7.28	282	125.7	1146	9.1

＜110〉Kazimierz Zimny Ces Co.,Ltd

＜120〉be used for concentrating the method for polypeptide

<130>PIDK0811587

<150>PA 2006 00488

<151>2006-04-04

<150>PA 2006 00922

<151>2006-07-05

<160>24

<170>FastSEQ for Windows Version 4.0

<210>1

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>1

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>2

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>2

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>3

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>3

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>4

<211>1034

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>4

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt cttcacccaa aatttgttgg gaagacccta gaaaccctgc 360

cagagaagag tgtggtggga accagctccc tgcgaagagc agcccagctg cagagaaagt 420

tcccgcatct ggagttcagg agtattcggg gaaacctcaa cacccggctt cggaagctgg 480

acgagcagca ggagttcagt gccatcatcc tggcaacagc tggcctgcag cgcatgggct 540

ggcacaaccg ggtggggcag atcctgcacc ctgaggaatg catgtatgct gtgggccagg 600

gggccttggg cgtggaagtg cgagccaagg accaggacat cttggatctg gtgggtgtgc 660

tgcacgatcc cgagactctg cttcgctgca tcgctgaaag ggccttcctg aggcacctgg 720

aaggaggctg cagtgtgcca gtagccgtgc atacagctat gaaggatggg caactgtacc 780

tgactggagg agtctggagt ctagacggct cagatagcat acaagagacc atgcaggcta 840

ccatccatgt ccctgcccag catgaagatg gccctgagga tgacccacag ttggtaggca 900

tcactgctcg taacattcca cgagggcccc agttggctgc ccagaacttg ggcatcagcc 960

tggccaactt gttgctgagc aaaggagcca aaaacatcct ggatgttgca cggcaattga 1020

acgatgccca ttaa 1034

<210>5

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>5

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacgggcagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaagg 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgtcatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caagggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>6

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>6

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggtt gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>7

<211>1034

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>7

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggct 840

accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaatta 1020

acgatgccca ttaa 1034

<210>8

<211>1035

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>8

atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca gacggacagt 60

gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat tgctatgtcc 120

accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa aagcctgttt 180

accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt tcactccttg 240

aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg caagcgggaa 300

aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct agaaaccctg 360

ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct gcagagaaag 420

ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct tcggaagctg 480

gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca gcgcatgggc 540

tggcacaacc gggtggggca gatcctgcac cctgaggaat gcatgtatgc tgtgggccag 600

ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct ggtgggtgtg 660

ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct gaggcacctg 720

gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg gcaactgtac 780

ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac catgcaggcc 840

accatccatg tccctaccca gcatgaagat ggccctgagg atgacccaca gttggtaggc 900

atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt gggcatcagc 960

ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc acggcaattg 1020

aacgatgccc attaa 1035

<210>9

<211>1260

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>9

cacaggaaac agctatgacc atgattacgc caagctcgaa attaaccctc actaaaggga 60

acaaaagctg gagctccacc gcggtggcgg ccgctctaga actagtggat cccccgggct 120

gcaggaattc atgagagtga ttcgcgtggg tacccgcaag agccagcttg ctcgcataca 180

gacggacagt gtggtggcaa cattgaaagc ctcgtaccct ggcctgcagt ttgaaatcat 240

tgctatgtcc accacagggg acaagattct tgatactgca ctctctaaga ttggagagaa 300

aagcctgttt accaaggagc ttgaacatgc cctggagaag aatgaagtgg acctggttgt 360

tcactccttg aaggacctgc ccactgtgct tcctcctggc ttcaccatcg gagccatctg 420

caagcgggaa aaccctcatg atgctgttgt ctttcaccca aaatttgttg ggaagaccct 480

agaaaccctg ccagagaaga gtgtggtggg aaccagctcc ctgcgaagag cagcccagct 540

gcagagaaag ttcccgcatc tggagttcag gagtattcgg ggaaacctca acacccggct 600

tcggaagctg gacgagcagc aggagttcag tgccatcatc ctggcaacag ctggcctgca 660

gcgcatgggc tggcacaacc gggttgggca gatcctgcac cctgaggaat gcatgtatgc 720

tgtgggccag ggggccttgg gcgtggaagt gcgagccaag gaccaggaca tcttggatct 780

ggtgggtgtg ctgcacgatc ccgagactct gcttcgctgc atcgctgaaa gggccttcct 840

gaggcacctg gaaggaggct gcagtgtgcc agtagccgtg catacagcta tgaaggatgg 900

gcaactgtac ctgactggag gagtctggag tctagacggc tcagatagca tacaagagac 960

catgcaggct accatccatg tccctgccca gcatgaagat ggccctgagg atgacccaca 1020

gttggtaggc atcactgctc gtaacattcc acgagggccc cagttggctg cccagaactt 1080

gggcatcagc ctggccaact tgttgctgag caaaggagcc aaaaacatcc tggatgttgc 1140

acggcaattg aacgatgccc attaataagc ttatcgatac cgtcgacctc gagggggggc 1200

ccggtaccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 1260

<210>10

<211>1113

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>10

cacacagcct actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60

gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct 120

cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctgg cctgcagttt 180

gaaatcattg ctatgtccac cacaggggac aagattcttg atactgcact ctctaagatt 240

ggagagaaaa gcctgtttac caaggagctt gaacatgccc tggagaagaa tgaagtggac 300

ctggttgttc actccttgaa ggacctgccc actgtgcttc ctcctggctt caccatcgga 360

gccatctgca agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420

aagaccctag aaaccctgcc agagaagagt gtggtgggaa ccagctccct gcgaagagca 480

gcccagctgc agagaaagtt cccgcatctg gagttcagga gtattcgggg aaacctcaac 540

acccggcttc ggaagctgga cgagcagcag gagttcagtg ccatcatcct ggcaacagct 600

ggcctgcagc gcatgggctg gcacaaccgg gttgggcaga tcctgcaccc tgaggaatgc 660

atgtatgctg tgggccaggg ggccttgggc gtggaagtgc gagccaagga ccaggacatc 720

ttggatctgg tgggtgtgct gcacgatccc gagactctgc ttcgctgcat cgctgaaagg 780

gccttcctga ggcacctgga aggaggctgc agtgtgccag tagccgtgca tacagctatg 840

aaggatgggc aactgtacct gactggagga gtctggagtc tagacggctc agatagcata 900

caagagacca tgcaggctac catccatgtc cctgcccagc atgaagatgg ccctgaggat 960

gacccacagt tggtaggcat cactgctcgt aacattccac gagggcccca gttggctgcc 1020

cagaacttgg gcatcagcct ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080

gatgttgcac ggcaattgaa cgatgcccat taa 1113

<210>11

<211>1380

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>11

agcaggtcct actatcgcct ccctctagtc tctgcttctt tggatccctg aggagggcag 60

aaggaagaaa acagcccaaa gatgagagtg attcgcgtgg gtacccgcaa gagccagctt 120

gctcgcatac agacggacag tgtggtggca acattgaaag cctcgtaccc tggcctgcag 180

tttgaaatca ttgctatgtc caccacaggg gacaagattc ttgatactgc actctctaag 240

attggagaga aaagcctgtt taccaaggag cttgaacatg ccctggagaa gaatgaagtg 300

gacctggttg ttcactcctt gaaggacctg cccactgtgc ttcctcctgg cttcaccatc 360

ggagccatct gcaagcggga aaaccctcat gatgctgttg tctttcaccc aaaatttgtt 420

gggaagaccc tagaaaccct gccagagaag agtgtggtgg gaaccagctc cctgcgaaga 480

gcagcccagc tgcagagaaa gttcccgcat ctggagttca ggagtattcg gggaaacctc 540

aacacccggc ttcggaagct ggacgagcag caggagttca gtgccatcat cctagcaaca 600

gctggcctgc agcgcatggg ctggcacaac cgggttgggc agatcctgca ccctgaggaa 660

tgcatgtatg ctgtgggcca gggggccttg ggcgtggaag tgcgagccaa ggaccaggac 720

atcttggatc tggtgggtgt gctgcacgat cccgagactc tgcttcgctg catcgctgaa 780

agggccttcc tgaggcacct ggaaggaggc tgcagtgtgc cagtagccgt gcatacagct 840

atgaaggatg ggcaactgta cctgactgga ggagtctgga gtctagacgg ctcagatagc 900

atacaagaga ccatgcaggc taccatccat gtccctgccc agcatgaaga tggccctgag 960

gatgacccac agttggtagg catcactgct cgtaacattc cacgagggcc ccagttggct 1020

gcccagaact tgggcatcag cctggccaac ttgttgctga gcaaaggagc caaaaccatc 1080

ctggatgttg cacggcagct taacgatgcc cattaactgg tttgtggggc acagatgcct 1140

gggttgctgc tgtccagtgc ctacatcccg ggcctcagtg ccccattctc actgctatct 1200

ggggagtgat taccccggga gactgaactg cagggttcaa gccttccagg gatttgcctc 1260

accttggggc cttgatgact gccttgcctc ctcagtatgt gggggcttca tctctttaga 1320

gaagtccaag caacagcctt tgaatgtaac caatcctact aataaaccag ttctgaaggt 1380

<210>12

<211>1377

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>12

cacacagcct actttccaag cggagccatg tctggtaacg gcaatgcggc tgcaacggcg 60

gaagaaaaca gcccaaagat gagagtgatt cgcgtgggta cccgcaagag ccagcttgct 120

cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct cgtaccctgg cctgcagttt 180

gaaatcattg ctatgtccac cacaggggac aagattcttg atactgcact ctctaagatt 240

ggagagaaaa gcctgtttac caaggagctt gaacatgccc tggagaagaa tgaagtggac 300

ctggttgttc actccttgaa ggacctgccc actgtgcttc ctcctggctt caccatcgga 360

gccatctgca agcgggaaaa ccctcatgat gctgttgtct ttcacccaaa atttgttggg 420

aagaccctag aaaccctgcc agagaagagt gtggtgggaa ccagctccct gcgaagagca 480

gcccagctgc agagaaagtt cccgcatctg gagttcagga gtattcgggg aaacctcaac 540

acccggcttc ggaagctgga cgagcagcag gagttcagtg ccatcatcct agcaacagct 600

ggcctgcagc gcatgggctg gcacaaccgg gtggggcaga tcctgcaccc tgagaaatgc 660

atgtatgctg tgggccaggg ggccttgggc gtggaagtgc gagccaagga ccaggacatc 720

ttggatctgg tgggtgtgct gcacgatccc gagactctgc ttcgctgcat cgctgaaagg 780

gccttcctga ggcacctgga aggaggctgc agtgtgccag tagccgtgca tacagctatg 840

aaggatgggc aactgtacct gactggagga gtctggagtc tagacggctc agatagcata 900

caagagacca tgcaggctac catccatgtc cctgcccagc atgaagatgg ccctgaggat 960

gacccacagt tggtaggcat cactgctcgt aacattccac gagggcccca gttggctgcc 1020

cagaacttgg gcatcagcct ggccaacttg ttgctgagca aaggagccaa aaacatcctg 1080

gatgttgcac ggcagcttaa cgatgcccat taactggttt gtggggcaca gatgcctggg 1140

ttgctgctgt ccagtgccta catcccgggc ctcagtgccc cattctcact gctatctggg 1200

gagtgattac cccgggagac tgaactgcag ggttcaagcc ttccagggat ttgcctcacc 1260

ttggggcctt gatgactgcc ttgcctcctc agtatgtggg ggcttcatct ctttagagaa 1320

gtccaagcaa cagcctttga atgtaaccaa tcctactaat aaaccagttc tgaaggt 1377

<210>13

<211>10024

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>13

aatcatgatt gttaattatg ttcatgatta caggcgcggt ggctcacgcc tgtactccca 60

gcactttggg aggccgaggt gggcgaatca cctgaggtca ggagttcaag acctgcctga 120

ctaacatgga gaaacctcat ctctaccaaa aatacaaaat tagccgggtg tggtggtgcg 180

tgcctgtaat cccagctact cggggggctg aggcaggaga attgcttgaa cccgggaggc 240

ggaggttgca gtgagctgag atcgtgccat tgcattccag cctgggcaac aagagcgaaa 300

ctccgtctca aaaaaaaaaa aaaattatgt tcatgggaaa gcacttttcc taacaagccc 360

ttttctcact acatgtaggt ttgtgctccc acttcagtta cttgtcttta ggcatgacct 420

ttaatctctc tgaaccagtt tcctcatttt aagaattgaa atgctggctg ggccagtcgt 480

cacgcctgta atcccagcac tttgggaggc caaggcgaga tgactgcttg agtccaggag 540

ttcgagacta gcctgggcaa catagtgagg ccacctcccc gctgtctcta taaaaaaatc 600

tagaaattag tcccacgtgg tgatgtgcgc ctgtagtccc agctgcttgg gaggctgagg 660

tggggggatc gctgaagccg ggaggtcaag gctgcagtga cccgtggtca tgccgctgca 720

ctctagtctg gggacacagt gagaccccgt atcaaaaaga aaaatgctgc ctatttcaag 780

gttgtagcaa agctaagttt gaacagagca aaggaagcgc catagaagct gcactacttg 840

ctcatgtcac agctggggaa tggggaggtc gaatggggag gtccactgtc gcaatgttcc 900

aattcccgcc cagagggagg gacctcccct tcgagggagg gcgccggaag tgacgcgagg 960

ctctgcggag accaggagtc agactgtagg acgacctcgg gtcccacgtg tccccggtac 1020

tcgccggccg gagcctccgg cttcccgggg ccgggggacc ttagcggcac ccacacacag 1080

cctactttcc aagcggagcc atgtctggta acggcaatgc ggctgcaacg gcggtgagtg 1140

ctgagccggt gaccagcaca ctttgggctt ctggacgagc cgtgcagcga ttggccccag 1200

gttgccatcc tcagtcgtct attggtcaga acggctatct tttttttttt tttttttttt 1260

tttttggtcc gagtagcttt taaagggcca gtagctcggt tgccctccgg aaggaatggg 1320

gaaatcagag agcggtgata ctgggttaag agtggaagga ttgtttggaa cggaactccg 1380

gtccctgcgg gcatctgggt gggattccca tcaggcctgg gatgcacggc tctagattta 1440

gtgacccaga ccaagaacgt tcgtctacac agacggggtc ctttcattcg aggctgggct 1500

gaggcggatg cagatacggc ccctttggga agacacgttc cacttttgat tcataggaga 1560

gagtatcagc caagcctccg aactgcacac aaacgtctta gaagtgcgcc ttctttttgt 1620

gttatagtgg tctcccagcc acagccaacg ctccaagtcc ccagctgtga cacacctact 1680

gaattactac cgtgggtggg aggccgccgt gggcctttcc attacgagcc tgcttgccga 1740

gccctgggct tgtgcacaga caaactgcag agctggtgga ggccactgcc aggccgagat 1800

aagaaagaga tggggagctg ctaatctccc cctgtccagc ctgttggtga gggctgggat 1860

ctttgctctt gcagtcattc cagagccctg gactaggagt aggaagatct gaattgtggc 1920

cccaactctc tttcggttat tagctctgtg accctaggca agtcacctca tcccttgatg 1980

ccacccgttg cttctgtaac atggtcccaa aggtgcctgt cttgtccacc tgataggatt 2040

tttgagacga caacaatatg caaaagcaat agcttcaaca tagaagtgct cagtgtttta 2100

ttttttaatg aaacggtttg acttggatat gctgtgcaca ttcaatgaac ttaaggaatt 2160

gtttgaacct agtagttctg ggaccttaga gtcctttctg tgggctccct gtggcccaga 2220

tttttggtgg ccacgtttaa tatcaagcct agcctaattt gcaaagggtc tcccagggtt 2280

aatttattgg agtgatcaca tggagtagac cagagtctga gggcagaaag ctgtcacctg 2340

cttcggcaat agaggcccca gatgtctggg tgcaaaagaa ctccatagca ccccgaccaa 2400

catggtgaaa ccccgtctct actaaaaata taaaaattag gccgagcaca gtggctcatg 2460

cctgtaatcc tagcactttg ggaggccgag gcaggtggat tgcctgagct caggagttcg 2520

agaccagcct agggaacaca gtgaaacccc gtttctacta aaaatacaaa aaattagccg 2580

acgtggtggc atgcgcctgc agtcccagct acttgggagg ctaagacagg agaatcgctt 2640

gaacctggga ggtggaggtt gcactgagcc gagaccgcgc cattgcactc cagcctgggt 2700

gacagagcgc aactccccct caaaaaaaga aaaaaatata tatatatata tatatatata 2760

tacacacata ttttagctgg gcatggtggt gtgcgtctgt agtagtccca gctacttggg 2820

aggctgagtc aggagaatcg cttgaacctg gaaggcagtg gttgtagtta gctgagaaca 2880

tgccactgca ctccagcctg ggcaacagag ggagactctg tctcaaaaaa aaaaaaaaaa 2940

aggaactaca taggatgaac atcccagatc agggaatgtt gactgtcgac agtatcagta 3000

tctacagtgg ctactgtctg atgtagaaag aaatgggatc aggctaggcg tggtggctca 3060

cgcctgtaat cccagctctt tgggaggctg gggcaggagg atcacaagtt cgagaccagc 3120

ctggccaaca cagtgaaacc ccgtctctac taaaaatgtg aaaattagct gggcatggtg 3180

gaacatgctg tagttccagc ttgaacccag gggtggaggt tgtagtgagc ctagatcacg 3240

ccactgcact ccagcctgag caaaacagtg agactctgtc taaaaaaaaa aaaaaaaaaa 3300

agagaaatgg gacctccgtc ttagactgaa gaattcagtt ctacgtgctt agcagtgaat 3360

acttttgtcc aaggtactct ggcaggagga agaggcgtgt cctcttgagt tcttgacttg 3420

ggctctggcc tgttaatatt tccatgttgg tgaaaccaga ggcagcactc taggtgaacg 3480

aactttaggc agcgcagcct cctagtctta tggaacatct gaggcagaag aaacctgagt 3540

ccaacctttt cattttatag atgaacaaac agatcctgat gggacagtgt acccaaggtc 3600

acccagccaa gaggctgagc aggactgtac gtcagatccg tttacctcag tccttaatgc 3660

atgcagtcca gccagattaa gggaccctta atactgtcag ctttccccac tgtgggatct 3720

tcatcctctt gacttctttt gtagccagac atctgggcct cttgctggag aaggtggcag 3780

cttgctgctc ttagactcta gtctactcca tgtggcatct ggatggcact gaaattttct 3840

caagtgcctt gtctgttgta gataatgaat ctatcctcca gtgactcagc acaggttccc 3900

cagtgtggtc ctggctgccc tgcccctgcc agctgcaggc cccacccttc ctgtggccag 3960

gctgatgggc cttatctctt tacccacctg gctgtgcaca gcactcccac tgacaactgc 4020

cttggtcaag gtgggcttca gggctcagtg tcctggttac tgcagcggca gcaacagcag 4080

gtcctactat cgcctccctc tagtctctgc ttctctggat ccctgaggag ggcagaaggt 4140

actgaggaag gttaaaggga ccagccttgg agtatttccc cactctgaga ctcagctggc 4200

cacaggccag gttctgaatt tcctttcttc caagccagtg attctggttc ttggacaagg 4260

tgttgaggaa cactagaaac agaggggact gtgacctggg gactttttct gcaggaagaa 4320

aacagcccaa agatgagagt gattcgcgtg ggtacccgca agagccaggt gggtgcagga 4380

gccggggtgg aggaggtttg tcagaacagt tatgatgctc acagcatcac aaattggggg 4440

actcagaggg ttagttccta gtatgaagga gatggggtgg ctgggcgtta agttccccgg 4500

gaaatggcag attacattct atggcaagat catccctagg ctgggaaaat tgttggagtg 4560

cagagggctc ccaagcccct tctcatgccc agatggaaat tccagtccct tcaggatctg 4620

cctaacctgt gacagtctaa agagtctgag ccgtggctgg gaagggcagg actaatccaa 4680

atctctaccc gcagcttgct cgcatacaga cggacagtgt ggtggcaaca ttgaaagcct 4740

cgtaccctgg cctgcagttt gaaatcagtg agttttctgg aaaggagtgg aagctaatgg 4800

gaagcccagt accccgagag gagagaacac aacatttctg gctttgccta tagctaaagc 4860

ccgtcccgct gccccgagat tccttctggg ctgctcccag ttctgaaggt gctttcctct 4920

gaatacctcc agctctgact acctggatta gcctggcatt taacatcttg agctttgggt 4980

ctttttatga gtgtttctgg tcttcctgct cgattgtata tactcagagg gcaggaacca 5040

gggattatgt gcctctgtcc ccatcatgaa tcgtagcaca gtgctaggct cagtaaatgc 5100

tgatcaataa tgagcacctg attgattgac tctctcctca gttgctatgt ccaccacagg 5160

ggacaagatt cttgatactg cactctctaa ggtaacaaca tcttcctccc cagttcttgt 5220

ccccactctt ctttccttcc ctgaagggat tcactcaggc tctttctgtc cggcagattg 5280

gagagaaaag cctgtttacc aaggagcttg aacatgccct ggagaagaat gagtaagtaa 5340

agataggaga gtgtggtgcc ctcccagtct cttgctggga ccctagtatg ctaggtctct 5400

tgctgggacc cggggtgtca gataggctgc tgggcttaaa ccctcagaga ggctgaaggc 5460

agctcatagg tgggtttttt caggcttcag aaaaggagag tgtctggttc tgagccatct 5520

ggctgcctgg actgcaagaa tggctggggg agggagggta ggagggagag taggagggag 5580

agtgagagga gagcagtttt catgctcctg agatcttgag aaggtgtgct tcctgaactg 5640

ccctaggctc caccactgaa gtagaggcag gggtgggtgg agaaggggtg aaggctggct 5700

gctcataccc tttctctttg cccccctctc ccatctctat agagtggacc tggttgttca 5760

ctccttgaag gacctgccca ctgtgcttcc tcctggcttc accatcggag ccatctgcaa 5820

gtaagagtct tgcaagtaag gggcttgggc aggggtaggc atcatgtgaa cctttgcctt 5880

tccctttggg gcctgaccct ctgcttcagg gttatctcct ctgccctgag gagtgttgac 5940

tggtggcaga aaactcaaga aataccagtg agttggcaat cgagagagaa tagaggtgat 6000

ctgaacttaa atctcttccc tcattctgtg cccttccctc ctcccccagg cgggaaaacc 6060

ctcatgatgc tgttgtcttt cacccaaaat ttgttgggaa gaccctagaa accctgccag 6120

agaagaggta agtggggcct ggataggcag cttggtggga tgtgcccaga agatgcaggg 6180

atgggaggag gaggaaagga acagtgactg cctagtgtta aaatctcatt gtaacttctc 6240

tctgggcagt gtggtgggaa ccagctccct gcgaagagca gcccagctgc agagaaagtt 6300

cccgcatctg gagttcagga gtattgtatc cttttagaag agtgacggat ccttttggaa 6360

gagtgacgga gacagcagcc aaggaaaaag acaaggtcta gagggctctg ggagtccgga 6420

gagtggaagg ggcttccagc aagcagcccg tggggtcagt ggcctgtctg tctttccatg 6480

cactcatccg tccactcatt tacagtctaa tgttttctta gccccagaca agtgttcaga 6540

gtgcaaggca ttggggataa tggtgagcaa gataaacatt cccctgcata tgtagagttt 6600

acgtcttact tagggataat gcagttatac tgaactgaat agtgactact tctggaggga 6660

tagggagtac ttcctttttt tttttttttt tttctgagac ggagtctcgc tctgttgccc 6720

aggttggagt gcagtggcgc aatctaggct cactgcaact tctgcctcct gagttcaagc 6780

aatcttcctg cctcagcctc ctaagtagtt gggattacag gtgccaccac acctggctaa 6840

tttttgtatt tttagtagag actgggtttc accatgttag tcaggctggt ctcaaactcc 6900

tgacctcagg tgatccacca gcctcggcct cccaaagggc tgggattaca ggcttgagcc 6960

ccgcacccgg tcagtacttc catttttata tgctactata ttgtcttgac ttttacaatg 7020

aatatgtagt acatttcata aaactaaatt taaaaatagt atgtgctaag tgctccaata 7080

agtgaagttg ggaattttct ggaaacttct agttggaaca tctaaacaca gaagtctggg 7140

gtgtcaggga aggtttctca gaggtcttgt aaccttggca agttatttag cctccctatg 7200

tcattttcct tatctgtaaa gtggggataa taatactacc ttcctcacag ggttgttgtg 7260

aagatgaaat gagctgacat atggaaagta cttttagagc agtgtctggc atgtagtaag 7320

tatgatgtaa ctgttagctg ttaacattaa gctgagagct ggaagatgac tgaaagtcag 7380

ccagctagag agggaaagac agactcaggc agagggaacc gcacgaggcc ccagattgcc 7440

cgacactgtg gtccttagca actctccaca gcggggaaac ctcaacaccc ggcttcggaa 7500

gatggacgag cagcaggagt tcagtgccat catcctggca acagctggcc tgcagcgcat 7560

gggctggcac aaccgggttg ggcaggtagg gcctgcccct atcctctccc cagctcatct 7620

gcatctcctt tctgccttac agtcatcccc aatttaggat ttttagactt tatgattgtg 7680

tgaaagcgat atacgttcag tagaaactgt acttagtacc catacagcca ttctgttttt 7740

tactttcagt acagtattca ttacatgaga tattcacttt attgtaaaac aggcttggtg 7800

tcagatgatt ttgtccaact ataataggct aatcttaagt gttctgagca catgtaaggt 7860

aggctaggtg tattaaatgc attttcagct tgttttcaac ttaacaatgg gtttatcagg 7920

atgtaaccct attgtaagtc aaggaccatc tgtcttcact tcttgaccac cccacctcta 7980

acaccgtagg ctgggaagat tgtgaatcag aggccagact ctaggctttc atggagaaaa 8040

tttacaaaaa aaaaaaaaag aggccagact cacacttagg cctacccagg ctttctagat 8100

gatagggaac tcccatctca ctgccaggtg cttttagaca cccccgtgtc cacccttttg 8160

actccctgtt ccgcctccac agatcctgca ccctgaggaa tgcatgtatg ctgtgggcca 8220

ggtacacttg accagggaag ccacatggtg acatatgcct tccctttgtt ctcaaccaag 8280

aagcttgtct cacaaccttc tgcatctgct tccccagaat agcattctca gggaggggca 8340

gaccttggga tgctaccggt ccaaaaggcg ctggggagca agtagataga ggtggtccca 8400

tgctttgcgc cattggttgg ggaaagatca ggcctgatgt cctaggatgt ttttccatca 8460

gggggccttg ggcgtggaag tgcgagccaa ggaccaggac atcttggatc tggtgggtgt 8520

gctgcacgat cccgagactc tgcttcgctg catcgctgaa agggccttcc tgaggcacct 8580

ggtagggcct gtgctccacc tgtggagggc tggggacttg gagagctggg aaaggtggca 8640

gggaagattt cttacatgaa tgctctgtat acagtgctaa ctcattcttg ttgaatgttg 8700

tgtatggata ggaccaggtc tgggcccaca gttgcctttt cagtgatgtc ctcaggtctg 8760

tggtcacagg gtggtgttaa gagcccttgc agctcacaag aacttcttgt tacaggaagg 8820

aggctgcagt gtgccagtag ccgtgcatac agctatgaag gatgggcaag taagtggggg 8880

gaaatgggcg ggaagccagg gaaaggagga ctgtggcatt tcttcctgtg catcccaggt 8940

ttctaggtag tcccctctca gactgtgctg aggcaactgt tttcttcccc agctgtacct 9000

gactggagga gtctggagtc tagacggctc agatagcata caagagacca tgcaggctac 9060

catccatgtc cctgcccagg taccaaagct ggagggcgag ggggtaataa acaagagtgc 9120

atataatctc ttgttctcac caaatcccac ctccttccct catacagcat gaagatggcc 9180

ctgaggatga cccacagttg gtaggcatca ctgctcgtaa cattccacga gggccccagt 9240

tggctgccca gaacttgggc atcagcctgg ccaacttgtt gctgagcaaa ggagccaaaa 9300

acatcctgga tgttgcacgg cagcttaacg atgcccatta actggtttgt ggggcacaga 9360

tgcctgggtt gctgctgtcc agtgcctaca tcccgggcct cagtgcccca ttctcactgc 9420

tatctgggga gtgattaccc cgggagactg aactgcaggg ttcaagcctt ccagggattt 9480

gcctcacctt ggggccttga tgactgcctt gcctcctcag tatgtggggg cttcatctct 9540

ttagagaagt ccaagcaaca gcctttgaat gtaaccaatc ctactaataa accagttctg 9600

aaggtgttgt gtgtgcgcgt gtggagttgg cgggaagata ggaacaaaca caaagccctt 9660

tcatccttac ctcagaggct gggacttttg cccagagttc tcctggtacg tcctttctgc 9720

ttctgcctca atagttttca tttcacacag aataaattgt ctcccaggaa caccaagaaa 9780

cagagccaca atcttaaatt cctatggttt gccccttcag ttaacagtag agcctgttta 9840

tattgcatgg cccctcccac ccctattatc aggaaagtat agaaagtcac taattctaca 9900

actctcttgc aaaatgaaaa caaatgctcc atttaaaaaa aaaacaatcc tttaataaaa 9960

ttagtccatc taaaactccc caatgcctaa ggttctagtc gtggaagggt tagctgcaga 10020

attc 10024

<210>14

<211>361

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>14

Met Ser Gly Asn Gly Asn Ala Ala Ala Thr Ala Glu Glu Asn Ser Pro

1 5 10 15

Lys Met Arg Val Ile Arg Val Gly Thr Arg Lys Ser Gln Leu Ala Arg

20 25 30

Ile Gln Thr Asp Ser Val Val Ala Thr Leu Lys Ala Ser Tyr Pro Gly

35 40 45

Leu Gln Phe Glu Ile Ile Ala Met Ser Thr Thr Gly Asp Lys Ile Leu

50 55 60

Asp Thr Ala Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu

65 70 75 80

Leu Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser

85 90 95

Leu Lys Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala

100 105 110

Ile Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys

115 120 125

Phe Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly

130 135 140

Thr Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro His

145 150 155 160

Leu Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg Leu Arg Lys

165 170 175

Met Asp Glu Gln Gln Glu Phe Ser Ala Ile Ile Leu Ala Thr Ala Gly

180 185 190

Leu Gln Arg Met Gly Trp His Asn Arg Val Gly Gln Ile Leu His Pro

195 200 205

Glu Glu Cys Met Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val

210 215 220

Arg Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp

225 230 235 240

Pro Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His

245 250 255

Leu Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr Ala Met Lys

260 265 270

Asp Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp Ser Leu Asp Gly Ser

275 280 285

Asp Ser Ile Gln Glu Thr Met Gln Ala Thr Ile His Val Pro Ala Gln

290 295 300

His Glu Asp Gly Pro Glu Asp Asp Pro Gln Leu Val Gly Ile Thr Ala

305 310 315 320

Arg Asn Ile Pro Arg Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile

325 330 335

Ser Leu Ala Asn Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp

340 345 350

Val Ala Arg Gln Leu Asn Asp Ala His

355 360

<210>15

<211>344

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>15

Met Arg Val Ile Arg Val Gly Thr Arg Lys Ser Gln Leu Ala Arg Ile

1 5 10 15

Gln Thr Asp Ser Val Val Ala Thr Leu Lys Ala Ser Tyr Pro Gly Leu

20 25 30

Gln Phe Glu Ile Ile Ala Met Ser Thr Thr Gly Asp Lys Ile Leu Asp

35 40 45

Thr Ala Leu Ser Lys Ile Gly Glu Lys Ser Leu Phe Thr Lys Glu Leu

50 55 60

Glu His Ala Leu Glu Lys Asn Glu Val Asp Leu Val Val His Ser Leu

65 70 75 80

Lys Asp Leu Pro Thr Val Leu Pro Pro Gly Phe Thr Ile Gly Ala Ile

85 90 95

Cys Lys Arg Glu Asn Pro His Asp Ala Val Val Phe His Pro Lys Phe

100 105 110

Val Gly Lys Thr Leu Glu Thr Leu Pro Glu Lys Ser Val Val Gly Thr

115 120 125

Ser Ser Leu Arg Arg Ala Ala Gln Leu Gln Arg Lys Phe Pro His Leu

130 135 140

Glu Phe Arg Ser Ile Arg Gly Asn Leu Asn Thr Arg Leu Arg Lys Met

145 150 155 160

Asp Glu Gln Gln Glu Phe Ser Ala Ile Ile Leu Ala Thr Ala Gly Leu

165 170 175

Gln Arg Met Gly Trp His Asn Arg Val Gly Gln Ile Leu His Pro Glu

180 185 190

Glu Cys Met Tyr Ala Val Gly Gln Gly Ala Leu Gly Val Glu Val Arg

195 200 205

Ala Lys Asp Gln Asp Ile Leu Asp Leu Val Gly Val Leu His Asp Pro

210 215 220

Glu Thr Leu Leu Arg Cys Ile Ala Glu Arg Ala Phe Leu Arg His Leu

225 230 235 240

Glu Gly Gly Cys Ser Val Pro Val Ala Val His Thr Ala Met Lys Asp

245 250 255

Gly Gln Leu Tyr Leu Thr Gly Gly Val Trp Ser Leu Asp Gly Ser Asp

260 265 270

Ser Ile Gln Glu Thr Met Gln Ala Thr Ile His Val Pro Ala Gln His

275 280 285

Glu Asp Gly Pro Glu Asp Asp Pro Gln Leu Val Gly Ile Thr Ala Arg

290 295 300

Asn Ile Pro Arg Gly Pro Gln Leu Ala Ala Gln Asn Leu Gly Ile Ser

305 310 315 320

Leu Ala Asn Leu Leu Leu Ser Lys Gly Ala Lys Asn Ile Leu Asp Val

325 330 335

Ala Arg Gln Leu Asn Asp Ala His

340

<210>16

<211>2022

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>16

ccggtaccgg ctcctcctgg gctccctcta gcgccttccc cccggcccga ctgcctggtc 60

agcgccaagt gacttacgcc cccgaccctg agcccggacc gctaggcgag gaggatcaga 120

tctccgctcg agaatctgaa ggtgccctgg tcctggagga gttccgtccc agccctgcgg 180

tctcccggta ctgctcgccc cggccctctg gagcttcagg aggcggccgt cagggtcggg 240

gagtatttgg gtccggggtc tcagggaagg gcggcgcctg ggtctgcggt atcggaaaga 300

gcctgctgga gccaagtagc cctccctctc ttgggacaga cccctcggtc ccatgtccat 360

gggggcaccg cggtccctcc tcctggccct ggctgctggc ctggccgttg cccgtccgcc 420

caacatcgtg ctgatctttg ccgacgacct cggctatggg gacctgggct gctatgggca 480

ccccagctct accactccca acctggacca gctggcggcg ggagggctgc ggttcacaga 540

cttctacgtg cctgtgtctc tgtgcacacc ctctagggcc gccctcctga ccggccggct 600

cccggttcgg atgggcatgt accctggcgt cctggtgccc agctcccggg ggggcctgcc 660

cctggaggag gtgaccgtgg ccgaagtcct ggctgcccga ggctacctca caggaatggc 720

cggcaagtgg caccttgggg tggggcctga gggggccttc ctgccccccc atcagggctt 780

ccatcgattt ctaggcatcc cgtactccca cgaccagggc ccctgccaga acctgacctg 840

cttcccgccg gccactcctt gcgacggtgg ctgtgaccag ggcctggtcc ccatcccact 900

gttggccaac ctgtccgtgg aggcgcagcc cccctggctg cccggactag aggcccgcta 960

catggctttc gcccatgacc tcatggccga cgcccagcgc caggatcgcc ccttcttcct 1020

gtactatgcc tctcaccaca cccactaccc tcagttcagt gggcagagct ttgcagagcg 1080

ttcaggccgc gggccatttg gggactccct gatggagctg gatgcagctg tggggaccct 1140

gatgacagcc ataggggacc tggggctgct tgaagagacg ctggtcatct tcactgcaga 1200

caatggacct gagaccatgc gtatgtcccg aggcggctgc tccggtctct tgcggtgtgg 1260

aaagggaacg acctacgagg gcggtgtccg agagcctgcc ttggccttct ggccaggtca 1320

tatcgctccc ggcgtgaccc acgagctggc cagctccctg gacctgctgc ctaccctggc 1380

agccctggct ggggccccac tgcccaatgt caccttggat ggctttgacc tcagccccct 1440

gctgctgggc acaggcaaga gccctcggca gtctctcttc ttctacccgt cctacccaga 1500

cgaggtccgt ggggtttttg ctgtgcggac tggaaagtac aaggctcact tcttcaccca 1560

gggctctgcc cacagtgata ccactgcaga ccctgcctgc cacgcctcca gctctctgac 1620

tgctcatgag cccccgctgc tctatgacct gtccaaggac cctggtgaga actacaacct 1680

gctggggggt gtggccgggg ccaccccaga ggtgctgcaa gccctgaaac agcttcagct 1740

gctcaaggcc cagttagacg cagctgtgac cttcggcccc agccaggtgg cccggggcga 1800

ggaccccgcc ctgcagatct gctgtcatcc tggctgcacc ccccgcccag cttgctgcca 1860

ttgcccagat ccccatgcct gagggcccct cggctggcct gggcatgtga tggctcctca 1920

ctgggagcct gtgggggagg ctcaggtgtc tggagggggt ttgtgcctga taacgtaata 1980

acaccagtgg agacttgcac atctgaaaaa aaaaaaaaaa aa 2022

<210>17

<211>1524

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>17

atgggggcac cgcggtccct cctcctggcc ctggctgctg gcctggccgt tgcacgtccg 60

cccaacatcg tgctgatctt tgccgacgac ctcggctatg gggacctggg ctgctatggg 120

caccccagct ctaccactcc caacctggac cagctggcgg cgggagggct gcggttcaca 180

gacttctacg tgcctgtgtc tctgtgcaca ccctctaggg ccgccctcct gaccggccgg 240

ctcccggttc ggatgggcat gtaccctggc gtcctggtgc ccagctcccg ggggggcctg 300

cccctggagg aggtgaccgt ggccgaagtc ctggctgccc gaggctacct cacaggaatg 360

gccggcaagt ggcaccttgg ggtggggcct gagggggcct tcctgccccc ccatcagggc 420

ttccatcgat ttctaggcat cccgtactcc cacgaccagg gcccctgcca gaacctgacc 480

tgcttcccgc cggccactcc ttgcgacggt ggctgtgacc agggcctggt ccccatccca 540

ctgttggcca acctgtccgt ggaggcgcag cccccctggc tgcccggact agaggcccgc 600

tacatggctt tcgcccatga cctcatggcc gacgcccagc gccaggatcg ccccttcttc 660

ctgtactatg cctctcacca cacccactac cctcagttca gtgggcagag ctttgcagag 720

cgttcaggcc gcgggccatt tggggactcc ctgatggagc tggatgcagc tgtggggacc 780

ctgatgacag ccatagggga cctggggctg cttgaagaga cgctggtcat cttcactgca 840

gacaatggac ctgagaccat gcgtatgtcc cgaggcggct gctccggtct cttgcggtgt 900

ggaaagggaa cgacctacga gggcggtgtc cgagagcctg ccttggcctt ctggccaggt 960

catatcgctc ccggcgtgac ccacgagctg gccagctccc tggacctgct gcctaccctg 1020

gcagccctgg ctggggcccc actgcccaat gtcaccttgg atggctttga cctcagcccc 1080

ctgctgctgg gcacaggcaa gagccctcgg cagtctctct tcttctaccc gtcctaccca 1140

gacgaggtcc gtggggtttt tgctgtgcgg actggaaagt acaaggctca cttcttcacc 1200

cagggctctg cccacagtga taccactgca gaccctgcct gccacgcctc cagctctctg 1260

actgctcatg agcccccgct gctctatgac ctgtccaagg accctggtga gaactacaac 1320

ctgctggggg gtgtggccgg ggccacccca gaggtgctgc aagccctgaa acagcttcag 1380

ctgctcaagg cccagttaga cgcagctgtg accttcggcc ccagccaggt ggcccggggc 1440

gaggaccccg ccctgcagat ctgctgtcat cctggctgca ccccccgccc agcttgctgc 1500

cattgcccag atccccatgc ctga 1524

<210>18

<211>507

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>18

Met Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala

1 5 10 15

Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly

20 25 30

Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn

35 40 45

Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val

50 55 60

Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg

65 70 75 80

Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser

85 90 95

Ara Gly Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala

100 105 110

Ala Arg Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val

115 120 125

Gly Pro Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe

130 135 140

Leu Gly Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr

145 150 155 160

Cys Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu

165 170 175

Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro

180 185 190

Trp Leu Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu

195 200 205

Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala

210 215 220

Ser His His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu

225 230 235 240

Arg Ser Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala

245 250 255

Ala Val Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu

260 265 270

Glu Thr Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg

275 280 285

Met Ser Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr

290 295 300

Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly

305 310 315 320

His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu

325 330 335

Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr

340 345 350

Leu Asp Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser

355 360 365

Pro Arg Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg

370 375 380

Gly Val Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr

385 390 395 400

Gln Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala

405 410 415

Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser

420 425 430

Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala

435 440 445

Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala

450 455 460

Gln Leu Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly

465 470 475 480

Glu Asp Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg

485 490 495

Pro Ala Cys Cys His Cys Pro Asp Pro His Ala

500 505

<210>19

<211>489

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<220>

＜221〉formylation

<222>51

＜223〉C-α formylglycine

<400>19

Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly

1 5 10 15

Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp

20 25 30

Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val

35 40 45

Ser Leu Xaa Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro

50 55 60

Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly

65 70 75 80

Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg

85 90 95

Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro

100 105 110

Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phe His Arg Phe Leu Gly

115 120 125

Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr Cys Phe

130 135 140

Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu Val Pro

145 150 155 160

Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro Trp Leu

165 170 175

Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu Met Ala

180 185 190

Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His

195 200 205

His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser

210 215 220

Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val

225 230 235 240

Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu Glu Thr

245 250 255

Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg Met Ser

260 265 270

Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr Thr Tyr

275 280 285

Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly His Ile

290 295 300

Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro

305 310 315 320

Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp

325 330 335

Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg

340 345 350

Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val

355 360 365

Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr Gln Gly

370 375 380

Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala Ser Ser

385 390 395 400

Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser Lys Asp

405 410 415

Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala Thr Pro

420 425 430

Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu

435 440 445

Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly Glu Asp

450 455 460

Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg Pro Ala

465 470 475 480

Cys Cys His Cys Pro Asp Pro His Ala

485

<210>20

<211>489

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>20

Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly Tyr Gly

1 5 10 15

Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro Asn Leu Asp

20 25 30

Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe Tyr Val Pro Val

35 40 45

Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro

50 55 60

Val Arg Met Gly Met Tyr Pro Gly Val Leu Val Pro Ser Ser Arg Gly

65 70 75 80

Gly Leu Pro Leu Glu Glu Val Thr Val Ala Glu Val Leu Ala Ala Arg

85 90 95

Gly Tyr Leu Thr Gly Met Ala Gly Lys Trp His Leu Gly Val Gly Pro

100 105 110

Glu Gly Ala Phe Leu Pro Pro His Gln Gly Phs His Arg Phe Leu Gly

115 120 125

Ile Pro Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr Cys Phe

130 135 140

Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu Val Pro

145 150 155 160

Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro Trp Leu

165 170 175

Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His Asp Leu Met Ala

180 185 190

Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe Leu Tyr Tyr Ala Ser His

195 200 205

His Thr His Tyr Pro Gln Phe Ser Gly Gln Ser Phe Ala Glu Arg Ser

210 215 220

Gly Arg Gly Pro Phe Gly Asp Ser Leu Met Glu Leu Asp Ala Ala Val

225 230 235 240

Gly Thr Leu Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu Glu Thr

245 250 255

Leu Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg Met Ser

260 265 270

Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr Thr Tyr

275 280 285

Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro Gly His Ile

290 295 300

Ala Pro Gly Val Thr His Glu Leu Ala Ser Ser Leu Asp Leu Leu Pro

305 310 315 320

Thr Leu Ala Ala Leu Ala Gly Ala Pro Leu Pro Asn Val Thr Leu Asp

325 330 335

Gly Phe Asp Leu Ser Pro Leu Leu Leu Gly Thr Gly Lys Ser Pro Arg

340 345 350

Gln Ser Leu Phe Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg Gly Val

355 360 365

Phe Ala Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr Gln Gly

370 375 380

Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala Ser Ser

385 390 395 400

Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser Lys Asp

405 410 415

Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala Gly Ala Thr Pro

420 425 430

Glu Val Leu Gln Ala Leu Lys Gln Leu Gln Leu Leu Lys Ala Gln Leu

435 440 445

Asp Ala Ala Val Thr Phe Gly Pro Ser Gln Val Ala Arg Gly Glu Asp

450 455 460

Pro Ala Leu Gln Ile Cys Cys His Pro Gly Cys Thr Pro Arg Pro Ala

465 470 475 480

Cys Cys His Cys Pro Asp Pro His Ala

485

<210>21

<211>1011

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>21

Met Gly Ala Tyr Ala Arg Ala Ser Gly Val Cys Ala Arg Gly Cys Leu

1 5 10 15

Asp Ser Ala Gly Pro Trp Thr Met Ser Arg Ala Leu Arg Pro Pro Leu

20 25 30

Pro Pro Leu Cys Phe Phe Leu Leu Leu Leu Ala Ala Ala Gly Ala Arg

35 40 45

Ala Gly Gly Tyr Glu Thr Cys Pro Thr Val Gln Pro Asn Met Leu Asn

50 55 60

Val His Leu Leu Pro His Thr His Asp Asp Val Gly Trp Leu Lys Thr

65 70 75 80

Val Asp Gln Tyr Phe Tyr Gly Ile Lys Asn Asp Ile Gln His Ala Gly

85 90 95

Val Gln Tyr Ile Leu Asp Ser Val Ile Scr Ala Leu Leu Ala Asp Pro

100 105 110

Thr Arg Arg Phe Ile Tyr Val Glu Ile Ala Phe Phe Ser Arg Trp Trp

115 120 125

His Gln Gln Thr Asn Ala Thr Gln Glu Val Val Arg Asp Leu Val Arg

130 135 140

Gln Gly Arg Leu Glu Phe Ala Asn Gly Gly Trp Val Met Asn Asp Glu

145 150 155 160

Ala Ala Thr His Tyr Gly Ala Ile Val Asp Gln Met Thr Leu Gly Leu

165 170 175

Arg Phe Leu Glu Asp Thr Phe Gly Asn Asp Gly Arg Pro Arg Val Ala

180 185 190

Trp His Ile Asp Pro Phe Gly His Ser Arg Glu Gln Ala Ser Leu Phe

195 200 205

Ala Gln Met Gly Phe Asp Gly Phe Phe Phe Gly Arg Leu Asp Tyr Gln

210 215 220

Asp Lys Trp Val Arg Met Gln Lys Leu Glu Met Glu Gln Val Trp Arg

225 230 235 240

Ala Ser Thr Ser Leu Lys Pro Pro Thr Ala Asp Leu Phe Thr Gly Val

245 250 255

Leu Pro Asn Gly Tyr Asn Pro Pro Arg Asn Leu Cys Trp Asp Val Leu

260 265 270

Cys Val Asp Gln Pro Leu Val Glu Asp Pro Arg Ser Pro Glu Tyr Asn

275 280 285

Ala Lys Glu Leu Val Asp Tyr Phe Leu Asn Val Ala Thr Ala Gln Gly

290 295 300

Arg Tyr Tyr Arg Thr Asn His Thr Val Met Thr Met Gly Ser Asp Phe

305 310 315 320

Gln Tyr Glu Asn Ala Asn Met Trp Phe Lys Asn Leu Asp Lys Leu Ile

325 330 335

Arg Leu Val Asn Ala Gln Gln Ala Lys Gly Ser Ser Val His Val Leu

340 345 350

Tyr Ser Thr Pro Ala Cys Tyr Leu Trp Glu Leu Asn Lys Ala Asn Leu

355 360 365

Thr Trp Ser Val Lys His Asp Asp Phe Phe Pro Tyr Ala Asp Gly Pro

370 375 380

His Gln Phe Trp Thr Gly Tyr Phe Ser Ser Arg Pro Ala Leu Lys Arg

385 390 395 400

Tyr Glu Arg Leu Ser Tyr Asn Phe Leu Gln Val Cys Asn Gln Leu Glu

405 410 415

Ala Leu Val Gly Leu Ala Ala Asn Val Gly Pro Tyr Gly Ser Gly Asp

420 425 430

Ser Ala Pro Leu Asn Glu Ala Met Ala Val Leu Gln His His Asp Ala

435 440 445

Val Ser Gly Thr Ser Arg Gln His Val Ala Asn Asp Tyr Ala Arg Gln

450 455 460

Leu Ala Ala Gly Trp Gly Pro Cys Glu Val Leu Leu Ser Asn Ala Leu

465 470 475 480

Ala Arg Leu Arg Gly Phe Lys Asp His Phe Thr Phe Cys Gln Gln Leu

485 490 495

Asn Ile Ser Ile Cys Pro Leu Ser Gln Thr Ala Ala Arg Phe Gln Val

500 505 510

Ile Val Tyr Asn Pro Leu Gly Arg Lys Val Asn Trp Met Val Arg Leu

515 520 525

Pro Val Ser Glu Gly Val Phe Val Val Lys Asp Pro Asn Gly Arg Thr

530 535 540

Val Pro Ser Asp Val Val Ile Phe Pro Ser Ser Asp Ser Gln Ala His

545 550 555 560

Pro Pro Glu Leu Leu Phe Ser Ala Ser Leu Pro Ala Leu Gly Phe Ser

565 570 575

Thr Tyr Ser Val Ala Gln Val Pro Arg Trp Lys Pro Gln Ala Arg Ala

580 585 590

Pro Gln Pro Ile Pro Arg Arg Ser Trp Ser Pro Ala Leu Thr Ile Glu

595 600 605

Asn Glu His Ile Arg Ala Thr Phe Asp Pro Asp Thr Gly Leu Leu Met

610 615 620

Glu Ile Met Asn Met Asn Gln Gln Leu Leu Leu Pro Val Arg Gln Thr

625 630 635 640

Phe Phe Trp Tyr Asn Ala Ser Ile Gly Asp Asn Glu Ser Asp Gln Ala

645 650 655

Ser Gly Ala Tyr Ile Phe Arg Pro Asn Gln Gln Lys Pro Leu Pro Val

660 665 670

Ser Arg Trp Ala Gln Ile His Leu Val Lys Thr Pro Leu Val Gln Glu

675 680 685

Val His Gln Asn Phe Ser Ala Trp Cys Ser Gln Val Val Arg Leu Tyr

690 695 700

Pro Gly Gln Arg His Leu Glu Leu Glu Trp Ser Val Gly Pro Ile Pro

705 710 715 720

Val Gly Asp Thr Trp Gly Lys Glu Val Ile Ser Arg Phe Asp Thr Pro

725 730 735

Leu Glu Thr Lys Gly Arg Phe Tyr Thr Asp Ser Asn Gly Arg Glu Ile

740 745 750

Leu Glu Arg Arg Arg Asp Tyr Arg Pro Thr Trp Lys Leu Asn Gln Thr

755 760 765

Glu Pro Val Ala Gly Asn Tyr Tyr Pro Val Asn Thr Arg Ile Tyr Ile

770 775 780

Thr Asp Gly Asn Met Gln Leu Thr Val Leu Thr Asp Arg Ser Gln Gly

785 790 795 800

Gly Ser Ser Leu Arg Asp Gly Ser Leu Glu Leu Met Val His Arg Arg

805 810 815

Leu Leu Lys Asp Asp Gly Arg Gly Val Ser Glu Pro Leu Met Glu Asn

820 825 830

Gly Ser Gly Ala Trp Val Arg Gly Arg His Leu Val Leu Leu Asp Thr

835 840 845

Ala Gln Ala Ala Ala Ala Gly His Arg Leu Leu Ala Glu Gln Glu Val

850 855 860

Leu Ala Pro Gln Val Val Leu Ala Pro Gly Gly Gly Ala Ala Tyr Asn

865 870 875 880

Leu Gly Ala Pro Pro Arg Thr Gln Phe Ser Gly Leu Arg Arg Asp Leu

885 890 895

Pro Pro Ser Val His Leu Leu Thr Leu Ala Ser Trp Gly Pro Glu Met

900 905 910

Val Leu Leu Arg Leu Glu His Gln Phe Ala Val Gly Glu Asp Ser Gly

915 920 925

Arg Asn Leu Ser Ala Pro Val Thr Leu Asn Leu Arg Asp Leu Phe Ser

930 935 940

Thr Phe Thr Ile Thr Arg Leu Gln Glu Thr Thr Leu Val Ala Asn Gln

945 950 955 960

Leu Arg Glu Ala Ala Ser Arg Leu Lys Trp Thr Thr Asn Thr Gly Pro

965 970 975

Thr Pro His Gln Thr Pro Tyr Gln Leu Asp Pro Ala Asn Ile Thr Leu

980 985 990

Glu Pro Met Glu Ile Arg Thr Phe Leu Ala Ser Val Gln Trp Lys Glu

995 1000 1005

Val Asp Gly

1010

<210>22

<211>8079

<212>DNA

＜213〉artificial sequence

<220>

＜223〉expression plasmid pLamanExp1

<400>22

agatcttcaa tattggccat tagccatatt attcattggt tatatagcat aaatcaatat 60

tggctattgg ccattgcata cgttgtatct atatcataat atgtacattt atattggctc 120

atgtccaata tgaccgccat gttggcattg attattgact agttattaat agtaatcaat 180

tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 240

tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 300

tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 360

aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt 420

caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc 480

tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca 540

gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat 600

tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa 660

caactgcgat cgcccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc 720

tatataagca gagctcgttt agtgaaccgt cagatcacta gaagctttat tgcggtagtt 780

tatcacagtt aaattgctaa cgcagtcagt gcttctgaca caacagtctc gaacttaagc 840

tgcagtgact ctcttaaggt agccttgcag aagttggtcg tgaggcactg ggcaggtaag 900

tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag 960

aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct 1020

ctccacaggt gtccactccc agttcaatta cagctcttaa ggctagagta cttaatacga 1080

ctcactatag gctagcctcg agaattcgcc gccatgggcg cctacgcgcg ggcttcgggg 1140

gtctgcgctc gaggctgcct ggactcagca ggcccctgga ccatgtcccg cgccctgcgg 1200

ccaccgctcc cgcctctctg ctttttcctt ttgttgctgg cggctgccgg tgctcgggcc 1260

gggggatacg agacatgccc cacagtgcag ccgaacatgc tgaacgtgca cctgctgcct 1320

cacacacatg atgacgtggg ctggctcaaa accgtggacc agtactttta tggaatcaag 1380

aatgacatcc agcacgccgg tgtgcagtac atcctggact cggtcatctc tgccttgctg 1440

gcagatccca cccgtcgctt catttacgtg gagattgcct tcttctcccg ttggtggcac 1500

cagcagacaa atgccacaca ggaagtcgtg cgagaccttg tgcgccaggg gcgcctggag 1560

ttcgccaatg gtggctgggt gatgaacgat gaggcagcca cccactacgg tgccatcgtg 1620

gaccagatga cacttgggct gcgctttctg gaggacacat ttggcaatga tgggcgaccc 1680

cgtgtggcct ggcacattga ccccttcggc cactctcggg agcaggcctc gctgtttgcg 1740

cagatgggct tcgacggctt cttctttggg cgccttgatt atcaagataa gtgggtacgg 1800

atgcagaagc tggagatgga gcaggtgtgg cgggccagca ccagcctgaa gcccccgacc 1860

gcggacctct tcactggtgt gcttcccaat ggttacaacc cgccaaggaa tctgtgctgg 1920

gatgtgctgt gtgtcgatca gccgctggtg gaggaccctc gcagccccga gtacaacgcc 1980

aaggagctgg tcgattactt cctaaatgtg gccactgccc agggccggta ttaccgcacc 2040

aaccacactg tgatgaccat gggctcggac ttccaatatg agaatgccaa catgtggttc 2100

aagaaccttg acaagctcat ccggctggta aatgcgcagc aggcaaaagg aagcagtgtc 2160

catgttctct actccacccc cgcttgttac ctctgggagc tgaacaaggc caacctcacc 2220

tggtcagtga aacatgacga cttcttccct tacgcggatg gcccccacca gttctggacc 2280

ggttactttt ccagtcggcc ggccctcaaa cgctacgagc gcctcagcta caacttcctg 2340

caggtgtgca accagctgga ggcgctggtg ggcctggcgg ccaacgtggg accctatggc 2400

tccggagaca gtgcacccct caatgaggcg atggctgtgc tccagcatca cgacgccgtc 2460

agcggcacct cccgccagca cgtggccaac gactacgcgc gccagcttgc ggcaggctgg 2520

gggccttgcg aggttcttct gagcaacgcg ctggcgcggc tcagaggctt caaagatcac 2580

ttcacctttt gccaacagct aaacatcagc atctgcccgc tcagccagac ggcggcgcgc 2640

ttccaggtca tcgtttataa tcccctgggg cggaaggtga attggatggt acggctgccg 2700

gtcagcgaag gcgttttcgt tgtgaaggac cccaatggca ggacagtgcc cagcgatgtg 2760

gtaatatttc ccagctcaga cagccaggcg caccctccgg agctgctgtt ctcagcctca 2820

ctgcccgccc tgggcttcag cacctattca gtagcccagg tgcctcgctg gaagccccag 2880

gcccgcgcac cacagcccat ccccagaaga tcctggtccc ctgctttaac catcgaaaat 2940

gagcacatcc gggcaacgtt tgatcctgac acagggctgt tgatggagat tatgaacatg 3000

aatcagcaac tcctgctgcc tgttcgccag accttcttct ggtacaacgc cagtataggt 3060

gacaacgaaa gtgaccaggc ctcaggtgcc tacatcttca gacccaacca acagaaaccg 3120

ctgcctgtga gccgctgggc tcagatccac ctggtgaaga cacccttggt gcaggaggtg 3180

caccagaact tctcagcttg gtgttcccag gtggttcgcc tgtacccagg acagcggcac 3240

ctggagctag agtggtcggt ggggccgata cctgtgggcg acacctgggg gaaggaggtc 3300

atcagccgtt ttgacacacc gctggagaca aagggacgct tctacacaga cagcaatggc 3360

cgggagatcc tggagaggag gcgggattat cgacccacct ggaaactgaa ccagacggag 3420

cccgtggcag gaaactacta tccagtcaac acccggattt acatcacgga tggaaacatg 3480

cagctgactg tgctgactga ccgctcccag gggggcagca gcctgagaga tggctcgctg 3540

gagctcatgg tgcaccgaag gctgctgaag gacgatggac gcggagtatc ggagccacta 3600

atggagaacg ggtcgggggc gtgggtgcga gggcgccacc tggtgctgct ggacacagcc 3660

caggctgcag ccgccggaca ccggctcctg gcggagcagg aggtcctggc ccctcaggtg 3720

gtgctggccc cgggtggcgg cgccgcctac aatctcgggg ctcctccgcg cacgcagttc 3780

tcagggctgc gcagggacct gccgccctcg gtgcacctgc tcacgctggc cagctggggc 3840

cccgaaatgg tgctgctgcg cttggagcac cagtttgccg taggagagga ttccggacgt 3900

aacctgagcg cccccgttac cttgaacttg agggacctgt tctccacctt caccatcacc 3960

cgcctgcagg agaccacgct ggtggccaac cagctccgcg aggcagcctc caggctcaag 4020

tggacaacaa acacaggccc cacaccccac caaactccgt accagctgga cccggccaac 4080

atcacgctgg aacccatgga aatccgcact ttcctggcct cagttcaatg gaaggaggtg 4140

gatggttagg tctgctggga tgggccctct agagtcgacc cgggcggccg cttcccttta 4200

gtgagggtta atgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac 4260

aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt 4320

tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt 4380

tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg 4440

taaaatccga taaggatcga tccgggctgg cgtaatagcg aagaggcccg caccgatcgc 4500

ccttcccaac agttgcgcag cctgaatggc gaatggacgc gccctgtagc ggcgcattaa 4560

gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4620

ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag 4680

ctctaaatcg ggggctccct ttagggttcc gatttagagc tttacggcac ctcgaccgca 4740

aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc 4800

gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa 4860

cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct 4920

attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt 4980

gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca 5040

tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 5100

gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 5160

tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 5220

ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 5280

gcttttttgg aggcctaggc ttttgcaaaa agctcccggg atggttcgac cattgaactg 5340

catcgtcgcc gtgtcccaaa atatggggat tggcaagaac ggagacctac cctggcctcc 5400

gctcaggaac gagttcaagt acttccaaag aatgaccaca acctcttcag tggaaggtaa 5460

acagaatctg gtgattatgg gtaggaaaac ctggttctcc attcctgaga agaatcgacc 5520

tttaaaggac agaattaata tagttctcag tagagaactc aaagaaccac cacgaggagc 5580

tcattttctt gccaaaagtt tggatgatgc cttaagactt attgaacaac cggaattggc 5640

aagtaaagta gacatggttt ggatagtcgg aggcagttct gtttaccagg aagccatgaa 5700

tcaaccaggc caccttagac tctttgtgac aaggatcatg caggaatttg aaagtgacac 5760

gtttttccca gaaattgatt tggggaaata taaacttctc ccagaatacc caggcgtcct 5820

ctctgaggtc caggaggaaa aaggcatcaa gtataagttt gaagtctacg agaagaaaga 5880

ctaattcgaa atgaccgacc aagcgacgcc caacctgcca tcacgatggc cgcaataaaa 5940

tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgaatcgata gcgataagga 6000

tccgcgtatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 6060

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 6120

acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 6180

cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 6240

taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 6300

tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 6360

aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 6420

ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 6480

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 6540

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 6600

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 6660

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 6720

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 6780

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 6840

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 6900

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6960

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 7020

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 7080

ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 7140

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 7200

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 7260

accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 7320

tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7380

tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7440

tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7500

cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7560

caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7620

cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7680

cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7740

gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7800

acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7860

atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7920

cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7980

gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 8040

tcctggcctt ttgctggcct tttgctcaca tggctcgac 8079

<210>23

<211>3761

<212>DNA

＜213〉homo sapiens (Homo Sapiens)

<400>23

ggctactctc ggcttcctgg caacgccgag cgaaagctat gactgcggcc gcgggttcgg 60

cgggccgcgc cgcggtgccc ttgctgctgt gtgcgctgct ggcgcccggc ggcgcgtacg 120

tgctcgacga ctccgacggg ctgggccggg agttcgacgg catcggcgcg gtcagcggcg 180

gcggggcaac ctcccgactt ctagtaaatt acccagagcc ctatcgttct cagatattgg 240

attatctctt taagccgaat tttggtgcct ctttgcatat tttaaaagtg gaaataggtg 300

gtgatgggca gacaacagac ggcactgagc cctcccacat gcattatgca ctagatgaga 360

attatttccg aggatacgag tggtggttga tgaaagaagc taagaagagg aatcccaata 420

ttacactcat tgggttgcca tggtcattcc ctggatggct gggaaaaggt ttcgactggc 480

cttatgtcaa tcttcagctg actgcctatt atgtcgtgac ctggattgtg ggcgccaagc 540

gttaccatga tttggacatt gattatattg gaatttggaa tgagaggtca tataatgcca 600

attatattaa gatattaaga aaaatgctga attatcaagg tctccagcga gtgaaaatca 660

tagcaagtga taatctctgg gagtccatct ctgcatccat gctccttgat gccgaactct 720

tcaaggtggt tgatgttata ggggctcatt atcctggaac ccattcagca aaagatgcaa 780

agttgactgg gaagaagctt tggtcttctg aagactttag cactttaaat agtgacatgg 840

gtgcaggctg ctggggtcgc attttaaatc agaattatat caatggctat atgacttcca 900

caatcgcatg gaatttagtg gctagttact atgaacagtt gccttatggg agatgcgggt 960

tgatgacggc ccaagagcca tggagtgggc actacgtggt agaatctcct gtctgggtat 1020

cagctcatac cactcagttt actcaacctg gctggtatta cctgaagaca gttggccatt 1080

tagagaaagg aggaagctac gtagctctga ctgatggctt agggaacctc accatcatca 1140

ttgaaaccat gagtcataaa cattctaagt gcatacggcc atttcttcct tatttcaatg 1200

tgtcacaaca atttgccacc tttgttctta agggatcttt tagtgaaata ccagagctac 1260

aggtatggta taccaaactt ggaaaaacat ccgaaagatt tctttttaag cagctggatt 1320

ctctatggct ccttgacagc gatggcagtt tcacactgag cctgcatgaa gatgagctgt 1380

tcacactcac cactctcacc actggtcgca aaggcagcta cccgcttcct ccaaaatccc 1440

agcccttccc aagtacctat aaggatgatt tcaatgttga ttacccattt tttagtgaag 1500

ctccaaactt tgctgatcaa actggtgtat ttgaatattt tacaaatatt gaagaccctg 1560

gcgagcatca cttcacgcta cgccaagttc tcaaccagag acccattacg tgggctgccg 1620

atgcatccaa cacaatcagt attataggag actacaactg gaccaatctg actataaagt 1680

gtgatgttta catagagacc cctgacacag gaggtgtgtt cattgcagga agagtaaata 1740

aaggtggtat tttgattaga agtgccagag gaattttctt ctggattttt gcaaatggat 1800

cttacagggt tacaggtgat ttagctggat ggattatata tgctttagga cgtgttgaag 1860

ttacagcaaa aaaatggtat acactcacgt taactattaa gggtcatttc gcctctggca 1920

tgctgaatga caagtctctg tggacagaca tccctgtgaa ttttccaaag aatggctggg 1980

ctgcaattgg aactcactcc tttgaatttg cacagtttga caactttctt gtggaagcca 2040

cacgctaata cttaacaggg catcatagaa tactctggat tttcttccct tctttttggt 2100

tttggttcag agccaattct tgtttcattg gaacagtata tgaggctttt gagactaaaa 2160

ataatgaaga gtaaaagggg agagaaattt atttttaatt taccctgtgg aagattttat 2220

tagaattaat tccaagggga aaactggtga atctttaaca ttacctggtg tgttccctaa 2280

cattcaaact gtgcattggc cataccctta ggagtggttt gagtagtaca gacctcgaag 2340

ccttgctgct aacactgagg tagctctctt catcttattt gcaagcggtc ctgtagatgg 2400

cagtaacttg atcatcactg agatgtattt atgcatgctg accgtgtgtc caagtgagcc 2460

agtgtcttca tcacaagatg atgctgccat aatagaaagc tgaagaacac tagaagtagc 2520

tttttgaaaa ccacttcaac ctgttatgct ttatgctcta aaaagtattt ttttattttc 2580

ctttttaaga tgatactttt gaaatgcagg atatgatgag tgggatgatt ttaaaaacgc 2640

ctctttaata aactacctct aacactattt ctgcggtaat agatattagc agattaattg 2700

ggttatttgc attatttaat ttttttgatt ccaagttttg gtcttgtaac cactataact 2760

ctctgtgaac gtttttccag gtggctggaa gaaggaagaa aacctgatat agccaatgct 2820

gttgtagtcg tttcctcagc ctcatctcac tgtgctgtgg tctgtcctca catgtgcact 2880

ggtaacagac tcacacagct gatgaatgct tttctctcct tatgtgtgga aggaggggag 2940

cacttagaca tttgctaact cccagaattg gatcatctcc taagatgtac ttacttttta 3000

aagtccaaat atgtttatat ttaaatatac gtgagcatgt tcatcatgtt gtatgattta 3060

tactaagcat taatgtggct ctatgtagca aatcagttat tcatgtaggt aaagtaaatc 3120

tagaattatt tataagaatt actcattgaa ctaattctac tatttaggaa tttataagag 3180

tctaacatag gcttagctac agtgaagttt tgcattgctt ttgaagacaa gaaaagtgct 3240

agaataaata agattacaga gaaaattttt tgttaaaacc aagtgatttc cagctgatgt 3300

atctaatatt ttttaaaaca aacattatag aggtgtaatt tatttacaat aaaatgttcc 3360

tactttaaat atacaattca gtgagttttg ataaattgat atacccatgt aaccaacact 3420

ccagtcaagc ttcagaatat ttccatcacc ccagaaggtt ctcttgtata cctgctcagt 3480

cagttccttt cactcccaat tgttggcagc cattgatagg aattctatca ctataggtta 3540

gttttctttg ttccagaaca tcatgaaagc ggcgtcatgt actgtgtatt cttatgaatg 3600

gtttctttcc atcagcataa tgatttgaga ttggtccatg ttgtgtgatt cagtggtttg 3660

ttccttctta tttctgaaga gttttccatt gtatgaatat accacaattt gtttcctccc 3720

caccagtttc tgatactaca attaaaactg tctacattta c 3761

<210>24

<211>669

<212>PRT

＜213〉homo sapiens (Homo Sapiens)

<400>24

Met Thr Ala Ala Ala Gly Ser Ala Gly Arg Ala Ala Val Pro Leu Leu

1 5 10 15

Leu Cys Ala Leu Leu Ala Pro Gly Gly Ala Tyr Val Leu Asp Asp Ser

20 25 30

Asp Gly Leu Gly Arg Glu Phe Asp Gly Ile Gly Ala Val Ser Gly Gly

35 40 45

Gly Ala Thr Ser Arg Leu Leu Val Asn Tyr Pro Glu Pro Tyr Arg Ser

50 55 60

Gln Ile Leu Asp Tyr Leu Phe Lys Pro Asn Phe Gly Ala Ser Leu His

65 70 75 80

Ile Leu Lys Val Glu Ile Gly Gly Asp Gly Gln Thr Thr Asp Gly Thr

85 90 95

Glu Pro Ser His Met His Tyr Ala Leu Asp Glu Asn Tyr Phe Arg Gly

100 105 110

Tyr Glu Trp Trp Leu Met Lys Glu Ala Lys Lys Arg Asn Pro Asn Ile

115 120 125

Thr Leu Ile Gly Leu Pro Trp Ser Phe Pro Gly Trp Leu Gly Lys Gly

130 135 140

Phe Asp Trp Pro Tyr Val Asn Leu Gln Lcu Thr Ala Tyr Tyr Val Val

145 150 155 160

Thr Trp Ile Val Gly Ala Lys Arg Tyr His Asp Leu Asp Ile Asp Tyr

165 170 175

Ile Gly Ile Trp Asn Glu Arg Ser Tyr Asn Ala Asn Tyr Ile Lys Ile

180 185 190

Leu Arg Lys Met Leu Asn Tyr Gln Gly Leu Gln Arg Val Lys Ile Ile

195 200 205

Ala Ser Asp Asn Leu Trp Glu Ser Ile Ser Ala Ser Met Leu Leu Asp

210 215 220

Ala Glu Leu Phe Lys Val Val Asp Val Ile Gly Ala His Tyr Pro Gly

225 230 235 240

Thr His Ser Ala Lys Asp Ala Lys Leu Thr Gly Lys Lys Leu Trp Ser

245 250 255

Ser Glu Asp Phe Ser Thr Leu Asn Ser Asp Met Gly Ala Gly Cys Trp

260 265 270

Gly Arg Ile Leu Asn Gln Asn Tyr Ile Asn Gly Tyr Met Thr Ser Thr

275 280 285

Ile Ala Trp Asn Leu Val Ala Ser Tyr Tyr Glu Gln Leu Pro Tyr Gly

290 295 300

Arg Cys Gly Leu Met Thr Ala Gln Glu Pro Trp Ser Gly His Tyr Val

305 310 315 320

Val Glu Ser Pro Val Trp Val Ser Ala His Thr Thr Gln Phe Thr Gln

325 330 335

Pro Gly Trp Tyr Tyr Leu Lys Thr Val Gly His Leu Glu Lys Gly Gly

340 345 350

Ser Tyr Val Ala Leu Thr Asp Gly Leu Gly Asn Leu Thr Ile Ile Ile

355 360 365

Glu Thr Met Ser His Lys His Ser Lys Cys Ile Arg Pro Phe Leu Pro

370 375 380

Tyr Phe Asn Val Ser Gln Gln Phe Ala Thr Phe Val Leu Lys Gly Ser

385 390 395 400

Phe Ser Glu Ile Pro Glu Leu Gln Val Trp Tyr Thr Lys Leu Gly Lys

405 410 415

Thr Ser Glu Arg Phe Leu Phe Lys Gln Leu Asp Ser Leu Trp Leu Leu

420 425 430

Asp Ser Asp Gly Ser Phe Thr Leu Ser Leu His Glu Asp Glu Leu Phe

435 440 445

Thr Leu Thr Thr Leu Thr Thr Gly Arg Lys Gly Ser Tyr Pro Leu Pro

450 455 460

Pro Lys Ser Gln Pro Phe Pro Ser Thr Tyr Lys Asp Asp Phe Asn Val

465 470 475 480

Asp Tyr Pro Phe Phe Ser Glu Ala Pro Asn Phe Ala Asp Gln Thr Gly

485 490 495

Val Phe Glu Tyr Phe Thr Asn Ile Glu Asp Pro Gly Glu His His Phe

500 505 510

Thr Leu Arg Gln Val Leu Asn Gln Arg Pro Ile Thr Trp Ala Ala Asp

515 520 525

Ala Ser Asn Thr Ile Ser Ile Ile Gly Asp Tyr Asn Trp Thr Asn Leu

530 535 540

Thr Ile Lys Cys Asp Val Tyr Ile Glu Thr Pro Asp Thr Gly Gly Val

545 550 555 560

Phe Ile Ala Gly Arg Val Asn Lys Gly Gly Ile Leu Ile Arg Ser Ala

565 570 575

Arg Gly Ile Phe Phe Trp Ile Phe Ala Asn Gly Ser Tyr Arg Val Thr

580 585 590

Gly Asp Leu Ala Gly Trp Ile Ile Tyr Ala Leu Gly Arg Val Glu Val

595 600 605

Thr Ala Lys Lys Trp Tyr Thr Leu Thr Leu Thr Ile Lys Gly His Phe

610 615 620

Ala Ser Gly Met Leu Asn Asp Lys Ser Leu Trp Thr Asp Ile Pro Val

625 630 635 640

Asn Phe Pro Lys Asn Gly Trp Ala Ala Ile Gly Thr His Ser Phe Glu

645 650 655

Phe Ala Gln Phe Asp Asn Phe Leu Val Glu Ala Thr Arg

660 665

<210>25

<211>11

<212>PRT

＜213〉artificial sequence

<220>

<223>11 residue basic peptide from HIV TAT protein

<400>25

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

1 5 10

<210>26

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉synthetic tat peptide

<400>26

Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala

1 5 10

Claims (28)

1. one kind concentrates the method for compositions that comprises polypeptide of interest, and it comprises:

A) centrifugal and/or filtration comprises the composition of polypeptide of interest;

B) concentrate supernatant liquor or the retentate that obtains from step a) respectively.

2. method according to claim 1, wherein, described polypeptide of interest is selected from the following group of forming: porphobilinogen deaminase, aryl sulphatase A, α mannosidase, galactocerebrosidase, and function equivalent part, and their analogue.

3. method according to claim 1, wherein, described polypeptide of interest comprises the amino acid that is selected from the following group of forming:

I) as SEQ ID NO.:14, SEQ ID NO.:15, SEQ ID NO.:18, SEQ ID NO.:19, SEQ ID NO.:20, SEQ ID NO.:21 and the defined arbitrary amino acid sequence of SEQ ID NO.:24;

Ii) as i) in the function equivalent part of definition aminoacid sequence; And

Iii) as i) or ii) in the function equivalent analogue of institute's definition aminoacid sequence, the aminoacid sequence of described analogue with as i) or ii) middle definition aminoacid sequence at least 75% identical.

4. method according to claim 1 and 2, wherein, step b) is undertaken by freeze-drying or evaporation.

5. according to any described method in the aforementioned claim, wherein, step b) is undertaken by ultrafiltration.

6. method according to claim 5, wherein, step b) is undertaken by tangential flow filtration.

7. method according to claim 5, wherein, step b) is carried out with centrifugal device.

8. according to any described method in the aforementioned claim, wherein, the described composition that comprises polypeptide of interest also comprises one or more compositions that are selected from the group of being made up of glycine, L-Serine, sucrose and N.F,USP MANNITOL.

9. according to any described method in the aforementioned claim, wherein, the described composition that comprises polypeptide of interest also comprises and is selected from by three (methylol) aminomethane hydrochloride, Trisodium Citrate and Na ₂HPO ₄One or more buffer reagents in the group of being formed.

10. according to any described method in the aforementioned claim, wherein, described method also comprises comprising from the step of the concentrate composition sterilization of polypeptide of interest that step b) obtains.

11. according to any described method in the aforementioned claim, wherein, described method comprises that also freeze-drying comprises the step of the concentrate composition of the polypeptide of interest that obtains from step b).

12. according to any described method in the aforementioned claim, wherein, centrifugal in the step a) carries out under 1800-2500g.

13. according to any described method in the aforementioned claim, wherein, being used for filtering filter in the step a), to have scope be the 0.20-5.0 micron pore size.

14. according to any described method in the aforementioned claim, wherein, described method also is included in the one or more the following steps before the step a):

I) recombinant expressed polypeptide of interest;

Ii) by one or more chromatographic step purifying polypeptide of interest; And

Iii) change the preparation damping fluid.

15. method according to claim 14 is wherein, at step I i) in chromatography be selected from the following group of forming: affinity chromatography, ion exchange chromatography and hydroxyapatite chromatography.

16. method according to claim 14, wherein, described method is included in the following step before the step a):

I) recombinant expressed polypeptide of interest;

Ii) make and comprise from step I) the composition of polypeptide of interest accept affinity chromatography; With

Iii) make and comprise step I i) the composition of polypeptide of interest accept ion exchange chromatography.

17. method according to claim 14, wherein, described method is included in the following step before the step a):

I) recombinant expressed polypeptide of interest

Ii) make and comprise from step I) the composition of polypeptide of interest accept affinity chromatography;

Iii) make and comprise from step I i) the composition of polypeptide of interest accept ion exchange chromatography; And

Iv) make the composition that comprises from step I polypeptide of interest ii) accept the hydroxyapatite column chromatography.

18. according to any described method in the claim 14, wherein, described method comprises to be utilized the recombinant expressed of nucleotide sequence, wherein said nucleotide sequence to comprise to be selected from the group that following sequence forms:

I) as SEQ ID NO.:1-13, SEQ ID NO.:16, SEQ ID NO.:17, SEQ ID NO.:22 and the defined any nucleotide sequence of SEQ ID NO.:23;

Ii) with as i) in the identical nucleotide sequence of definition nucleotide sequence at least 75%.

19. according to any described method in claim 16 or 17, wherein, described method comprises that also dilution or diafiltration comprise from step I i) step of the composition of the polypeptide of interest that obtains.

20. comprise the composition of 10mg/ml polypeptide at least, described polypeptide is selected from the following group of forming: porphobilinogen deaminase, aryl sulphatase A, lysosome alpha-Mannosidase and galactocerebrosidase.

21. composition according to claim 20, wherein, described polypeptide is the polypeptide as definition in claim 3.

Be used for the purposes of subcutaneous injection 22. comprise the composition that 75-250mg/ml is selected from the polypeptide in the group of being made up of porphobilinogen deaminase, aryl sulphatase A, lysosome alpha-Mannosidase and galactocerebrosidase in preparation to mammiferous medicine.

23. purposes according to claim 22, wherein, described polypeptide is the polypeptide as definition in claim 3.

24. according to claim 22 or 23 described purposes, wherein, described medicine is used for the treatment of acute intermittent porphyria, metachromatic leukodystrophy, lysosomal storage disease α-mannosidosis or Krabbe disease.

25. the method for treatment Mammals acute intermittent porphyria, it comprises that subcutaneous injection contains the composition of 500-300mg/ml porphobilinogen deaminase.

26. the method for treatment Mammals metachromatic leukodystrophy, it comprises that subcutaneous injection contains the composition of 50-300mg/ml aryl sulphatase A.

27. the method for treatment Mammals lysosomal storage disease α-mannosidosis, it comprises the composition of subcutaneous injection 50-300mg/ml lysosome alpha-Mannosidase.

28. the method for treatment Mammals Krabbe disease, it comprises that subcutaneous injection contains the composition of 50-300mg/ml galactocerebrosidase.