CN1547610A - DNA for achieving high expression of growth hormone and use thereof - Google Patents
DNA for achieving high expression of growth hormone and use thereof Download PDFInfo
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- CN1547610A CN1547610A CNA028166523A CN02816652A CN1547610A CN 1547610 A CN1547610 A CN 1547610A CN A028166523 A CNA028166523 A CN A028166523A CN 02816652 A CN02816652 A CN 02816652A CN 1547610 A CN1547610 A CN 1547610A
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
A DNA encoding a fused protein wherein a leader peptide Leader consisting of one or more amino acid residues from the N-terminus of a cell wall protein CWP of a bacillus, an amino acid sequence Cleavage to be used in enzymatic cleavage and an amino acid sequence GH of growth hormone, its functional fragment, variant or analog are appropriately ligated to nH consisting of a plural number of His residues and a linker Linker. Another DNA further containing a DNA sequence enabling the expression of the above DNA, a vector containing this DNA allowing the expression, host cells containing this vector and a process for producing a recombinant growth hormone by using these host cells are further disclosed.
Description
Technical field
The present invention relates to a kind of fusion rotein coded DNA, this fusion rotein contains and has the tethelin that specific regulating comprises the physiologically active of human Mammals growth and morphogenesis, or identical with its function, perhaps have further protein fragments or the analogue that improves function.Relate in addition and use this kind DNA, express a large amount of needed tethelin or its functional protein fragment, varient or analogues of producing by it.
Background technology
Tethelin is by a kind of peptide hormone of anterior lobe of hypophysis excretory, is the indispensable material of physical growth in vegetative period, particularly bone growth.If can not secrete tethelin for a certain reason, growth will stop, thereby causes nanism.
Medical tethelin can be used for the treatment of this nanism.Tethelin not only can be treated nanism, also is applicable to the cretinism that various diseases causes, and just in broadened application to effectively preventing many-sides such as aging.
Medical tethelin of past is to extract from the pituitary body of corpse, thereby available amount rareness and price is expensive high.The gene recombination technology that the 1970's set up also is used to produce tethelin, and the 1980's were set up the method with bacillus coli gene recombinant production tethelin.(putting down into 6 years-No. 65318, publication referring to Japanese patent gazette puts down into 7 years-No. 46988 communiques, patent gazettes and puts down into 8 years-No. 2314)
With the escherichia coli expression exogenous protein generally is to express (the flat 6-65318 communique of Japanese patent gazette) in tenuigenin, or it is secreted in the pericentral siphon.(publication is put down into 7 years-No. 46988, patent gazette and is put down into 8 years-No. 2314), but last class methods exist and form inclusion body often and sex change maybe can not form for keeping the very important problems such as disulfide linkage of physiological activity of protein; Back one class methods have solved the problem of the folding and disulfide linkage in protein space, but the excretory amount is restricted in periplasmic space.For this reason, present situation is to have the protein of disulfide linkage also to be restricted with this class of intestinal bacteria great expression tethelin.
Developed system in recent years with bacillus pumilus (Bacillus brevis) expressing gene recombinant protein, utilize this system, can be at activated state, the following polypeptide (human epithelial cell multiplicaiton factor) with disulfide linkage of expressing of state that promptly normally forms disulfide linkage also is secreted in the substratum in a large number.(No. the 2082727th, Japanese patent gazette, clear and 62 years-No. 201583 communiques of publication, Yamagata, H.et al., J.Bacteriol.169,1239-1245 (1987), Pelican Gao Chongsan, Japan's chemurgy meeting will, 61,669-676 (1987), Takao, M.et al., Appl.Microbiol.Biotechnol., 30,75-80 (1989), Yamagata, H.et al., Proc.Natl.Acad.Sci.USA, 86,3589-3593 (1989)), this a large amount of generations have the proteinic system of disulfide linkage to be subjected to people's attention.
Bacillus pumilus expression system is secreted the principle of exogenous protein in substratum, be owing to be right after the foreign protein mass-energy inducement signal peptide permeate through cell membranes of cell wall protein signal peptide, by signal peptidase exogenous protein and signal peptide cutting are come then, justacrine is in substratum.The human epithelial cell multiplicaiton factor utilizes this system to realize that great expression and secretion are good examples, but not all exogenous protein can both utilize this system's great expression secretion.As the report that utilizes this expression system expression-secretion human growth hormone, have and be right after human growth hormone in the back of signal peptide and make it to be secreted into report (Japanese publication is put down into communique 7 years-No. 51072) in the substratum, also promising raising secretory volume follows signal peptide closely with the form of fused protein and connects and make it excretory report (Japanese publication is put down into communique 11 years-No. 341991).About a kind of phraseology in back, though can see the high expression level of fusion rotein, but, because convenience for the aminoacid sequence discerned, be used for the N-terminal of tethelin that the proteolytic enzyme that the cutting of fusion rotein and tethelin is come is cut out many Gly or Ser, become the varient of tethelin, so do not obtain the spontaneous growth hormone.In addition, even the production of relative growth hormonal variations body also wishes to obtain further to raise the efficiency the technology that produces a large amount of desired proteins.
Therefore, the purpose of this invention is to provide the signal peptide that utilizes cell wall protein, the aminoacid sequence of suitably selecting to be used for a large amount of secreting, expressing tethelin is formed the fusion rotein that is also connected and obtain, and can further improve the fused protein of growth hormone secretion amount in host cell.Simultaneously, the enzyme that the objective of the invention is to select the tethelin the same with natural product that can obtain not contain the additional amino acid sequence in the fusion rotein of expression is cut the position, makes the combination that contains each aminoacid sequence of discovery under the situation that cutting do not reduce with the secretory volume of the fusion rotein of aminoacid sequence.
Content of the present invention
The inventor has realized that successfully the great expression secretion contains bioactive tethelin of possessing of disulfide linkage or its functional fragment, contains the fusion rotein of varient or analogue.Method is to use the cell wall protein (CWP of bacillus separately, Cell Wall Proteins) a kind of intermediate cell wall-held protein (MWP in, Middle Wall Protein) N-terminal the 1st to the 20th amino acids sequence (leading aminoacid sequence, Leader), or, dispose tethelin after being right after cutting part (Cleavage) Ile-Glu-Gly-Arg of enzyme or Glu-Asn-Leu-Tyr-Phe-Gln-Gly sequence with a plurality of His residues or as the Asp-Tyr-Asp-Ile-Pro-Thr-Thr of joint (Linker).Developed and used the enzyme that to discern above-mentioned cutting part to act on this fusion rotein, thereby be able to mass production natural type tethelin (20kd, 22kd), perhaps obtained the N-terminal technology of the variation thing of the tethelin of residual Gly (20kd, 22kd) still.
First embodiment of the present invention provides and is the fusion rotein coded DNA, this fusion rotein comprises the following aminoacid sequence that is linked in sequence: from bacillus cell wall-held protein matter (cwp) N-terminal, the leading peptide of forming by an above amino-acid residue (Leader), at the hit aminoacid sequence (GH) of aminoacid sequence (Cleavage), tethelin or its functional fragment, tethelin varient or its analogue that utilize of enzyme, and the aminoacid sequence of Cleavage is an Ile-Glu-Gly-Arg (sequence numbering: 1).
In other embodiments of the present invention, the DNA of encoding fusion protein is provided, this fusion rotein comprises the following aminoacid sequence that is linked in sequence: from bacillus cell wall-held protein matter (CWP) N-terminal, the leading peptide of forming by an above amino-acid residue (Leader), at the hit aminoacid sequence (GH) of aminoacid sequence (Cleavage), tethelin or its functional fragment, tethelin varient or the analogue that utilize of enzyme, and GH is 20kd human growth hormone (GH 20kd).
The DNA of the fusion rotein that also providing in other embodiment of the present invention encodes is formed by connecting in the following order, promptly from bacillus cell wall-held protein matter (CWP) N-terminal, the leading peptide of forming by an above amino-acid residue (Leader), the nH that a plurality of histidine residues is formed, at enzyme the hit aminoacid sequence (Cleavage) that utilizes and the aminoacid sequence (GH) of tethelin or its functional fragment, tethelin varient or analogue.In preferred embodiments, be 6 histidine residues.
In the preferred embodiment of the present invention, GH is the human growth hormone (GH22kd) of 22kd or the human growth hormone of 20kd; 1) or Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cleavage2, sequence numbering: 2) restriction endonuclease recognition sequence (Cleavage) is that (Cleavage 1, sequence numbering: for Ile-Glu-Gly-Arg.
In addition, also promising specific fusion proteins coded DNA, this specific fusion proteins is to connect the joint of being made up of 1 above amino-acid residue to form in the restriction endonuclease recognition sequence upstream, and the aminoacid sequence of best joint is an Asp-Tyr-Asp-Ile-Pro-Thr-Thr (sequence numbering: 3).
Cell wall protein is with intermediate cell wall-held protein (MWP) the best, and leading peptide is then special to be good with N-terminal the 1st to the 20th amino acids sequence from MWP.
In the other embodiments of the present invention, DNA is provided, this DNA be in 5 ' the terminal upstream of the DNA of the above-mentioned fusion rotein of coding by the DNA that contains the necessary for gene expression promoter sequence (Promoter), being linked in sequence with this at the DNA (CWPsp) of the signal peptide of the SD sequence in promotor 3 ' terminal downstream and coding bacillus cell wall-held protein matter (CWP) forms.Promotor used herein is preferably from bacillus cell wall-held protein matter (CWP).
In other embodiments of the present invention, the invention provides carrier, it contains DNA, and this DNA is linked in sequence with this by the DNA (CWPsp) of the signal peptide of the SD sequence in the DNA that contains the necessary for gene expression promoter sequence (Promoter), promotor 3 ' terminal downstream and coding bacillus cell wall-held protein matter (CWP) in 5 ' the terminal upstream of the DNA of the above-mentioned fusion rotein of coding to form.
Moreover, the invention provides the host cell that utilizes above-mentioned carrier to carry out conversion or transfection.Proper host cell is a bacillus, and this belongs in the bacterium best with bacillus pumilus (Bacillus brevis).
In the particularly preferred embodiment of the present invention, the host cell that contains above-mentioned carrier is a bacterial strain, declaring of this bacterial strain is numbered FERM BP-7727, declares registration in September 7 calendar year 2001 to the biological trust center of Independent Administrative Leged Industrial Technology Complex Inst's patent (the international associated agency of budapest treaty mandate) that is positioned at the 6th in a kind of ground of raised path between farm fields 1 fourth order, east, つ く ば city, Hitachinaka County, Japan, 1 central authorities.
The invention provides the production method of tethelin or its functional fragment, varient or analogue, wherein cultivate above-mentioned host cell, fusion rotein is expressed on the substratum of cultivating this host cell, obtain tethelin or its functional fragment, varient or analogue with the enzyme patterning method from this fusion rotein cutting, produce recombinant human growth hormone or its functional fragment, varient or analogue.
Description of drawings
The recombinate sketch map of the expression vector (pNU211R2L5) in the bacillus pumilus of Fig. 1 syzygy MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd.
Fig. 2 represents the collection of illustrative plates in the SDS-PAGE gel electrophoresis of substratum of various transformants.
The 1st swimming lane: labelled protein;
The 2nd swimming lane: negative control (the plasmid pNU211R2L5 that does not contain foreign protein);
The 3rd swimming lane: MWPsp-GH22kd;
The 4th swimming lane: MWPsp-MWP
Mp20-Cleavage 1-GH22kd;
The 5th swimming lane: MWPsp-MWPmp20-joint-Cleavage 1-GH22kd;
The 6th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd;
The 7th swimming lane: MWPsp-MWPmp20-joint-Cleavage 1-GH20kd;
The 8th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd;
The 9th swimming lane: MWPsp-MWPmp20-joint-Cleavage 2-GH22kd;
The 10th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd;
Fig. 3 represents the protein immunoblotting collection of illustrative plates of various transformants
The 1st swimming lane: negative control (the plasmid pNU211R2L5 that does not contain foreign protein);
The 2nd swimming lane: MWPsp-GH22kd;
The 3rd swimming lane: MWPsp-MWPmp20-Cleavage 1-GH22kd;
The 4th swimming lane: MWPsp-MWPmp20-joint-Cleavage 1-GH22kd;
The 5th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd;
The 6th swimming lane: MWPsp-MWPmp20-joint-Cleavage 1-GH20kd;
The 7th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd;
The 8th swimming lane: MWPsp-MWPmp20-joint-Cleavage 2-GH22kd;
The 9th swimming lane: MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd;
Fig. 4 is the figure of the quantitative values that obtains of the high performance liquid chromatography (HPLC) of expression by various transformant excretory human growth hormones,
A post: negative control (the plasmid pNU211R2L5 that does not contain foreign protein);
B post: MWPsp-GH22kd;
C post: MWPsp-MWPmp20-Cleavage 1-GH22kd;
D post: MWPsp-MWPmp20-joint-Cleavage 1-GH22kd;
E post: MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd;
Fig. 5 is the figure of high performance liquid chromatography (HPLC) wash-out of the various human growth hormones of expression
A figure: positive control (Norditropin);
B figure: natural GH22kd;
C figure: N-terminal has glycine (Gly) anomaly GH22kd;
D figure: natural type GH20kd;
Fig. 6 be the expression various human growth hormones cause put on weight the effect figure (data: mean value ± SD,
*: P＜0.01)
Among the figure ● expression negative control [0.25% bovine serum albumin (BSA)/0.9%NaCl], zero expression positive control (Norditropin), △ represents that N-terminal has the anomaly GH22kd of glycine, and ◇ represents natural type GH 22kd, and represents natural type GH 20kd.
Fig. 7 be the increase bone length effect that causes of the various human growth hormones of expression figure (data: mean value ± SD,
*: P＜0.05,
*: P＜0.01)
A post: negative control [0.25% bovine serum albumin (BSA)/0.9%NaCl]
B post: positive control (Norditropin)
The C post: N-terminal has the anomaly GH 22kd of Gly
D post: natural type GH 22kd tethelin
E post: natural type GH 20kd
The best mode that carries out an invention
According to the present invention, might when significantly improve bioactive tethelin of the performance that contains disulfide linkage or its functional fragment, varient or analogue output, go back mass production and natural product and have the tethelin of same N-terminal sequence and the recombinant chou of functional fragment thereof.
As the cell wall protein (cwp) in the fusion rotein of being included in as leading peptide, for example can adopt from strain of i (bacillus) pumilus 47 (FERM P-7224: clear and 60 years-No. 58074 communiques of Japanese publication, clear and 62 years-No. 201589 communiques of publication) intermediate cell wall-held protein [MWP, middle wall protein, see J.Bacteriol., 169:1239-1245 (1989), sequence numbering 40], HWP[S-layer proteins from bacterial strain HPD31 (FERM BP-1087: Japanese publication is put down into communique 4 years-No. 278091), in cell walls, form hexagonal array, see J.Bacteriol., 172:1312-1320 (1990), sequence numbering 41], but be not limited to the above.According to embodiment of the present invention, suitable leading peptide is the partial sequence of MWP.Preferred leading peptide, it is the aminoterminal amino-acid residue of the mature protein of MWP, report as put down into 11 years-No. 341991 communiques in Japanese publication,, just can see the effect that improves the growth hormone secretion amount of expressing by at 1 above amino-acid residue of N-terminal assembling.In an embodiment of the present invention, and amino terminal amino acid residue number can greater or less than 20.
Cleavage may be subjected to the aminoacid sequence that enzyme is cut, as long as the tethelin that its downstream connects is not subjected to enzyme to cut the influence of processing, does not then limit the sequence of its cutting especially.According to one embodiment of the invention, the suitable aminoacid sequence that enzyme is cut is the Ile-Glu-Gly-Arg (sequence numbering 1) that can be discerned by factor Xa, utilize this sequence, enzyme is cut the N-terminal of handling the resulting GH in back and is contained unnecessary amino-acid residue hardly, therefore preferred this sequence.The present invention can also utilize restriction endonuclease recognition sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly (sequence numbering 2), and this sequence can be discerned by TEV proteolytic enzyme.In addition, No. 5179007 communique of United States Patent (USP) pointed out, can be also had Glu-Asn-Leu-Try-Phe-Gln-Ser (sequence numbering 4) by the aminoacid sequence that TEV proteolytic enzyme is discerned, if adopt sequence 2 or sequence 4, the N-terminal of the tethelin after enzyme is cut processing contains glycine or serine residue, but the present invention confirms that N-terminal has the tethelin of glycine residue, and its biological activity is identical with natural product.
Though the optimal tethelin of the present invention is human growth hormone,,, then not necessarily must be human growth hormone if be expected to occur high expression level secretion effect as long as can be assembled on the part of tethelin with fusion rotein form of the present invention.That is, the mammiferous tethelin except that the people, or the tethelin of other biological also can.These tethelin comprise those functions and regulate growth or morphogenesis in vivo, for example relevant with metabolism with the promoting bone growth spontaneous growth hormone that physiologically active is arranged is identical, perhaps has to improve the function fragment or the analogue of (comprise and suppress detrimental action).So-called human growth hormone hHG (sequence numbering 38) is that people's pituitary body frontal lobe produces and the excretory peptide hormone.It is by the GH-1 genes encoding, and sophisticated molecule is that the intramolecularly that obtains behind the signal peptide that the processing excision is made up of 26 amino-acid residues has two disulfide linkage, by the molecule that 191 amino-acid residues are formed, and the about 22kd of molecular weight.Its known varient is that sophisticated molecule also is made up of 191 amino-acid residues by the hHG-V (sequence numbering 39) of gene GH-2 coding, the about 22kd of molecular weight, but it is not at pituitary body, and in placenta, express.13 amino-acid residues are different in the ripe molecule of hHG and hHG-V, but the two all has the physiologically active of tethelin.Thereby, in the tethelin of the present invention, comprise also and have only part to change in those 13 amino acid, or become similarly other amino acid that expection will show the varient or the analogue of tethelin physiologically active with these amino acid from hHG.Simultaneously, find that the 32nd to 46 amino-acid residue of hHG and hHG-V lacks, molecular weight is 20kd, and the molecule of they and 22kd is the same also to have the effect that equal promotion is grown, but it is all lower to urge the activity of glycosuria effect and insulin action.When only considering to promote growth result,, also can consider to use these that fragment of disappearance has taken place according to patient's state of health.Therefore the present invention also should comprise functional fragment, varient and analogue with corresponding physiologically active except that the tethelin that comprises tethelin (GH) with fusion protein form expression, natural type.
For example, be the human growth hormone coded DNA, can utilize the test kit of the people's pituitary body mRNA that buys and synthesizing single-stranded cDNA to obtain.If according to known dna sequence dna, utilize the commercially available synthetic short chain DNA of DNA synthetic primer that is used for, generally can be by polymerase chain reaction (PCR) the needed dna fragmentation that increases.Utilize pcr amplification, working cycle comprise the DNA sex change, with the annealing of primer, and the prolongation of chain obtains the DNA amount that needs through 20 circulations.
About joint, be and leading peptide-nH (Leader-nH) or the aminoacid sequence that is connected with leading peptide, Cleavage-GH can improve the secretory volume of fused protein whereby, or guarantee the efficient that enzyme is cut.This aminoacid sequence is not special qualification, as long as satisfy above-mentioned functions.But, according to embodiment of the present invention, particularly preferred aminoacid sequence is Asp-Tyr-Asp-Ile-Pro-Thr-Thr (sequence numbering 3), even the partial amino-acid residue of this sequence is by other amino-acid substitutions, as long as keep the effect of high secretory volume, also can use such constant series.
There is the effect that increases the expressing fusion protein secretory volume that contains tethelin the leading peptide back followed by a plurality of Histidines (His) residue of assembling.A plurality of histidine residues in the assembling of the front and back of foreign protein, be used as the marker of high efficiency separation purifying foreign protein, but inventor's discovery has unexpectedly obtained to increase the effect of the fusion rotein secretory volume that contains tethelin by importing this aminoacid sequence always.According to embodiment of the present invention appropriate H is residue number is 6, but is not limited in this, also can adopt the amino acid that is made of a plurality of histidine residues.
The DNA of the fusion rotein that coding provided by the invention is formed by connecting the sequence of above elaboration by suitable order can make up this field and make for the technology that the public grasps.
Each dna sequence dna that for example prepares integral part by chemosynthesis or clone's method one by one connects these DNA sequence with ligase enzyme then, in conjunction with adopting PCR method to produce needed DNA.The embodiment that specifically please refer to the application is understood.At Maniatis T.et al., Molecular Cloning 2
NdEd., A Laboratory Manual, Cold Spring HarborLaboratory (1989); Innis M.A.et al., PCR Protocols, A Guide to Methodsand Applications, the general technology of publication also can adopt among the Academic Press (1990) etc.
In other embodiments of the present invention, in order in host cell, to express above-mentioned fusion rotein, a carrier is provided, this carrier contains DNA, and this DNA is linked in sequence with this by the DNA that contains the necessary promoter sequence of genetic expression (Promoter), at the SD sequence in this promotor 3 ' terminal downstream and bacillus cell wall-held protein (cwp) at the DNA 5 ' upstream termination of this fusion rotein of coding to form.
According to embodiment of the present invention, suitable promotor is the promotor of the CWP of bacillus.As CWP, the same with description for Leader, can adopt MWP, or, not have strict restriction from the HWP of bacterial strain HPD31 (FERMBP-1087) from strain of i (bacillus) pumilus 47 (FERM P-7224).The promotor of bacillus, the Spac promotor of regulating effect has been arranged, from the promotors of φ 105 phages, and from the amylase of host cell structure gene or proteinic promotor or the like [Molecular BiologicalMethods for Bacillus, Harwood, C.R.﹠amp; Cutting, S.M.eds., pp202-203 (1990)], but preferably select those can make the promotor of the genetic expression of 3 ' terminal downstream connection.For example the promotor of MWP and HWP can be fit to use the transcribing of putting down in writing separately from sequence number 40 and 41 of 5 ' untranslated section existence to begin the interior dna sequence dna of selecting of promotor scope that position 1～5 exists.
The SD sequence that promotor 3 ' terminal downstream connects, known be the base sequence that is rich in pyrimidine (referring to Shine J. and Dalgarno, L., Eur.J.biochem.1:57 (1), pp.221-230), preferably select to promote to form the sequence of protein synthesis complex body.The proper sequence that adopts among the present invention is AGAGGAGGAGA (sequence numbering 42, SD2) or the GAAAGGAGGTGA (sequence numbering 43, SD1) that is present in the MWP translation starting point upstream of bacillus pumilus.
In addition, by the DNA (CWPsp) of the signal peptide of the cell wall protein (CWP) of coding bacillus being connected to DNA N-terminal (5 ' end) upstream of the above-mentioned fusion rotein of coding, after translating into association's arrival cytolemma of proteinic signal peptide and fusion rotein formation, fusion rotein will be separated from association owing to the effect of signal peptidase, and justacrine is to the extracellular.The signal peptide of MWP and HWP equally can be as CWPsp, and the aminoacid sequence of the signal peptide of MWP is particularly suitable for using.
The present invention also provides the carrier of the DNA that comprises the invention described above.The carrier that can use must meet following condition at least, promptly has to be inserted into suitable insertion position to DNA provided by the invention, just has the cleavage site of restriction enzyme; This DNA can be at host cell inner expression; And can be in host cell self-replicating etc.Carrier can contain replication origin and terminator sequence, also can contain the selective marker with drug resistant gene, nutritional requirement complementary gene etc.The plasmid that carrier of the present invention preferably can duplicate in bacillus.PNU200 for example, pHY500[Proc.Natl.Acad.Sci.USA, 86:pp.3589-3593 (1989)], pHY4831[J.Bacteriol., 169:pp1239-1245 (1987)], pNU100 (Appl.Microbiol, Biotechnol., 30:pp 75-80 (1989)), pNU211, [J.Biochem., 112:pp.488-491 (1992)], pNU211R2L5 (Japanese publication is put down into communique 7 years-No. 170984), pHY700 (Japanese publication is put down into communique 4 years-No. 278091), pHT210 (Japanese publication is put down into communique 6 years-No. 133782), pHT110R2L5[Appl.Microiol.Biotechnol., (1994)] etc., be exactly these but do not limit 30:pp.358-363 all can be used as carrier.In concrete example of the present invention,, can make expression vector pNU-hGH by building mode as shown in Figure 1.
The present invention further provides the host cell that comprises the bacillus that transforms with above-mentioned carrier.Bacillus have strain of i (bacillus) pumilus 47 (FERM P-7224: Japanese publication clear with 60 years-No. 58074 communiques, publication is clear and 62 years-No. 201589 communiques), strain of i (bacillus) pumilus 47K (Japanese publication is put down into communique 2 years-No. 257876), strain of i (bacillus) pumilus 310K (Japanese publication is put down into communique 6 years-No. 296485) and strain of i (bacillus) pumilus HPD31 (FERM BP-1087: Japanese publication is put down into communique 4 years-No. 278091) etc., but be not limited to these.Expression vector pNU-hGH is transferred to the recombinant bacteria that obtains among the strain of i (bacillus) pumilus 47-5Q, declare registration (original trust) to the biological trust center of Independent Administrative Leged Industrial Technology Complex Inst's patent (the international associated agency of budapest treaty mandate) that is positioned at the 6th in a kind of ground of raised path between farm fields 1 fourth order, east, つ く ば city, Hitachinaka County, Japan, 1 central authorities in September 7 calendar year 2001.Declaring of this bacterial strain is numbered FERM BP-7727.
With electroporation [Methods in Enzymol., (1993)] etc. 217:pp.23-33 method has been transferred to the above-mentioned expression vector that is obtained in the host cell of bacillus etc., under the condition that can express, with this bacterium of suitable culture medium culturing, can contain the fusion rotein of tethelin at the thalline external secretion.
Method separation and purification from substratum such as after finishing yeast culture, fusion rotein can the applied in any combination gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatogram, electrophoresis, isoelectric focusing electrophoresis.Carry out enzyme by this fusion rotein and cut processing separation and purification, can cut out GH, promptly natural type tethelin (20kd, 22kd), or N-terminal has the anomaly tethelin (22kd) of glycine residue (Gly), or functional fragment, varient or analogue.The recombinant human growth hormone of Huo Deing has kept and has kept the necessary disulfide linkage of biological activity like this, can directly be used as pharmaceuticals.
Carry out enzyme and cut processing, just carried out much less according to the sequence that has imported Cleavage in fusion rotein, the enzyme that employing can be cut on Cleavage is implemented also can under the optimum condition of this enzyme reaction.For example in the fusion rotein that imports numbering 1 sequence, under about 4～about 25 ℃, about 7～about 9 the condition of pH, make the Xa factor reaction, then in the fusion rotein that imports numbering 2 or 3 sequences, under about 4～about 37 ℃, about 7～about 9 the condition of pH, make proteolytic enzyme TEV reaction, can realize that enzyme cuts processing.
Therefore, the invention provides the method for mass production recombinant human growth hormone or its functional fragment, varient or analogue.This method is by cultivating the bacillus that transforms etc. by above-mentioned expression vector, and it is expressed in growth medium, cuts out from fusion rotein with enzyme process that tethelin or its functional fragment, varient or analogue realize again.
Embodiment
Specific embodiment of the present invention below is described, but these embodiment are not limited to the present invention.
During the DNA of preparation encoding fusion protein, adopt the method that the dna fragmentation by PCR (polymerase chain reaction) amplification is coupled together by the dna ligase reaction.In the present embodiment, MWPsp is meant the signal peptide of MWP, and MWPmp20 then is meant 20 amino acid that the proteic N-terminal of ripe MWP begins.
In embodiment 1, put down in writing the fusion dna that has connected fusion rotein MWPmp20-(His) 6-joint-Cleavage 1-GH 22kd on the preparation MWPsp (being equivalent to 20 amino acid that the C-terminal of MWP signal peptide begins), the method that promptly prepares MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd, and the method that makes up the carrier that inserts this material, the producer rule of other fused proteins is only summarized.
Inserted MWPsp-MWPmp20-(His) 6-joint-Cleavage1-GH22kd fusion dna
The structure of carrier (phGH)
(1) acquisition of dna fragmentation MWPsp-MWPmp20
The a template DNA
With known method [Molecular Cloning 2
NdEd., A Laboratory Manual, Cold Spring Harbor Laboratory (1989)] go out the 840ng genomic dna as template DNA from bacillus pumilus (Bacillus brevis47-50 strain) extracting.
The b primer
According to Yamagata, H. etc., J.Bacteriol., 169, pp.1239-1245 (1987)] and Tsuboi, A. etc., J.Bacteriol., 170, pp.935-945 (1988)] definite MWP base sequence, the following primer of organic synthesis:
Forward primer: 5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering: 5, ultimate density 0.1 μ M)
Reverse primer: 5 '-TACGGTTTTTTCCATATCAGC-3 ' (sequence numbering: 6, ultimate density 0.1 μ M)
C TaqDNA polysaccharase
Commercial goods (GIBCO BRL company product) 5U
D other
Tris-HCl (ultimate density 20mM, pH8), MgCl
2(ultimate density 2.5mM), dNTPs (dATP, dGTP, dCTP, dTTP, every kind ultimate density is 50 μ M)
Experimental procedure
Get each 100 μ l of above-mentioned a～d reagent in the test tube of 0.5ml, press known method [Innis, M.A. etc., PCR Protocols, A guide to methods and applications, AcademicPress (1990)] carry out the PCR reaction (each round-robin condition is: sex change: 94 ℃ 1 minute, annealing: 53 ℃ 1 minute, the prolongation of DNA chain: 72 ℃ 1 minute, 30 circulations).After the PCR reaction finished, behind the phenol concentration of reaction solution, the sepharose with 0.8% is electrophoresis under conditions suitable.The Urtrafree C3H that produces with MILLIPORE company receives PCR product, i.e. dna fragmentation together from sepharose
MWPsp-MWPmp20.
The PCR product that reclaims with ethanol sedimentation and vacuum-drying, is dissolved in an amount of distilled water after the phenol extracting, carries out blunt end reaction (use precious wine to make the DNA leveling test kit that company produces, method is according to appended specification sheets).
(2) acquisition of dna fragmentation (His) 6-joint-Cleavage 1
According to genetic code dictionary [Molecular Cloning 2
NdEd., A Laboratory Manual, Cold Spring Harbor Laboratory (1989)] aminoacid sequence (forward oligonucleotide sequence 5 '-CATCATCATCATCATCACGACTATGATATCCCGACCACTATCGAAGGTCGT-3 ' (sequence numbering 8) of His-His-His-His-His-His-Asp-Tyr-Asp-Ile-Pro-Thr-Thr-Ile-Glu-Gly-Arg (sequence numbering 7) of chemical synthesis coding (His) 6-joint-Cleavage 1, reverse oligonucleotide sequence 5 '-ACGACCTTCGATAGTGGTCGGGATATCATAGTCGTGATGATGATGATGATG-3 ' (sequence numbering 9), phosphorylation reaction is (with ニ Star Port Application ジ-Application company product polynueleotide kinase, the by specification operation) after the reaction, at 10mM Tris-HCl (pH8), 5mM MgCl
2Handled 5 minutes 37 ℃ of annealing in 15 minutes in the solution at 95 ℃.Annealing back double chain DNA fragment TEV with the phenol extracting after, with ethanol sedimentation, after the vacuum-drying in an amount of many distilled water of solution.
(3) acquisition of dna fragmentation, human growth hormone GH22kd
Except that following step, obtain terminal smooth dna fragmentation by (1) as much step
GH22kd
◆ with having inserted the plasmid vector of dna fragmentation GH22kd as template DNA
The plasmid vector that has inserted GH22kd obtains as follows
With the strand cDNA synthetic agent box of PHARMACIA company product, synthesize people's pituitary body cDNA from commercially available people's pituitary body mRNA (production of CLONTECH company) according to appended specification sheets.
With this cDNA is template, according to Roskam, W.G.et al.[Nucleic Acids Res., 7:pp.305-320 (1979)] and Martial, J.A.et al.[Science, 205, pp.602-607 (1979)] definite human growth hormone gene sequence, the primer of synthetic forward primer [5 '-ATGGCTACAGGCTCCCGGAC-3 ' (sequence numbering 10)] and reverse primer [5 '-CTAGAAGCCACAGCTGCCCT-3 ' sequence numbering 11)], (condition is 94 ℃ of each circulations 1 minute to carry out PCR reaction, annealing: 55 ℃ 1 minute, 72 ℃ 1 minute, 35 circulations) obtaining the PCR product, is human growth hormone (hGH) DNA with this PCR product, clones on pGEM-T carrier (Promega company product).
◆ use the forward primer of 5 '-TTCCCAACCATTCCCTTATC-3 ' (sequence numbering 12), the reverse primer of 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11) is as primer.
◆ the reaction conditions of PCR is set at 25 circulations, and each circulation is for sex change: 94 ℃ 1 minute, annealing: 55 ℃ 1 minute, the DNA chain extension: 72 ℃ 30 seconds.
(4) acquisition of MWPsp-MWPmp20-(His) 6-joint-Cleavage 1 fusion dna
Except that following step, terminal smooth fusion dna, MWPsp-MWPmp20-(His) 6-joint-Cleavage 1 have similarly been obtained by above-mentioned (1)
◆ dna fragmentation (His) 6-joint-Cleavage 1 that dna fragmentation MWPsp-MWPmp20 that obtains with step (1) and step (2) obtain respectively gets in right amount and is mixed, and the products that obtained in 30 minutes 16 ℃ of reactions with dna ligation kit (precious wine is produced product) are as template DNA.
◆ with the reverse primer of 5 '-ACGACCTTCGATAGTGGTCGG-3 ' (sequence numbering 13) as PCR reaction for the first time.
◆ for the first time the reaction conditions of PCR is: sex change in each circulation: 94 ℃ 1 minute, annealing: 53 ℃ 1 minute, 72 ℃ of the prolongations of DNA chain 1 minute, 25 circulations.
Then with the ニ Star Port Application polynueleotide kinase that ジ-Application company produces, the by specification operation is carried out phosphorylation to the PCR reaction product.The PCR product of phosphorylation is produced the dna ligation kit of product after restriction enzyme HinII cutting with precious wine, be inserted on the carrier (STRATAGENE company produces, Blue Scrip SK-), with known method [Molecular Cloning 2
NdEd., A Laboratory Manual, Cold Spring HarborLaboratory (1989)] transformed into escherichia coli DH5 α bacterial strain, from transformant, purify as the plasmid DNA of carrier.Employing is used for determining the forward primer (M13 forward primer) of carrier base sequence or the base sequence that reverse primer (M13 reverse primer) determines it, confirms to have formed MWPsp-MWPmp20-(His) 6-joint-Cleavage1 fusion dna.
Then, with the carrier that inserted MWPsp-MWPmp20-(His) 6-joint-Cleavage 1 fusion dna as template, the employing forward primer [5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering 5)] and reverse primer [5 '-ACGACCTTCGATAGTGGTCGG-3 ' (sequence numbering 13)], carry out the 2nd PCR reaction under above-mentioned same condition, the fusion dna that obtains blunt end is MWPsp-MWPmp20-(His) 6-joint-Cleavage 1.
(5) having inserted MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22Kd merges
The acquisition of the carrier of DNA
Except that following step, the step same by above-mentioned (4) obtains fusion dna, promptly inserted the carrier (phGH) of MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd
◆ the fusion dna that step (4) is obtained,
MWPsp-MWPmp20-(His) 6-joint- Cleavage 1 HeThe dna fragmentation GH22kd that step (3) obtains respectively gets in right amount and is mixed, and the products that obtained in 30 minutes 16 ℃ of reactions with dna ligation kit (precious wine is produced product) are as template DNA.
◆ adopt 5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering 5) to make forward primer and 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11) and make reverse primer as primer.
◆ the reaction conditions of PCR is: sex change in each circulation: 94 ℃ 1 minute, anneal 53 ℃ 1 minute, 72 ℃ of the prolongations of DNA chain 1 minute, 25 circulations.
Inserted the carrier of MWPsp-MWPmp20-joint-Cleavage 1-GH22kd fusion dna
Structure
With embodiment 1 resulting fusion dna is that MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd is a template, forward primer with 5 '-GACTATGATATCCCGACCACT-3 ' (sequence numbering 14), the reverse primer of 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11), amplification of DNA fragments joint-Cleavage 1-GH22kd.Then by the combination ligation put down in writing of embodiment 1 and PCR reaction, will obtain inserting the carrier of fusion dna MWPsp-MWPmp20-joint-Cleavage 1-GH22kd by the dna fragmentation of the dna fragmentation MWPsp-MWPmp20 of embodiment 1 (1) gained and joint-Cleavage 1-GH22kd.
Inserted the structure of the carrier of MWPsp-MWPmp20-Cleavage 1-GH22kd fusion dna
Build
With embodiment 1 resulting fusion dna is that MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd is a template, forward primer with 5 '-ATCGAAGGTCGTTTCCCAACC-3 ' (sequence numbering 15), the reverse primer of 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11), dna fragmentation Cleavage 1-GH22kd has increased.Ligation and PCR reaction by combination embodiment 1 record then obtained inserting the carrier of fusion dna MWPsp-MWPmp20-Cleavage 1-GH22kd by the dna fragmentation of the dna fragmentation MWPsp-MWPmp20 of embodiment 1 (1) gained and Cleavage 1-GH22kd.
Inserted MWPsp-MWPmp20-(His) 6-Joint-
Cleavage 1-GH20kd fusion dna The structure of carrier
With embodiment 1 resulting fusion dna is that MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd is a template, forward primer with 5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering 5), reverse primer with 5 '-AAACTCCTGGTAGGTGTCAAA-3 ' (sequence numbering 16), amplification of DNA fragments MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH (A), also use the forward primer of 5 '-AACCCCCAGACCTCCCTCTGT-3 ' (sequence numbering 17), with the reverse primer of 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11), amplification of DNA fragments GH (B).Ligation and the PCR that is put down in writing by combination embodiment 1 reacts then, obtained inserting the carrier of fusion dna MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd by these two dna fragmentations.
Inserted MWPsp-MWPmp20-joint-Cleavage 1-GH20kd fusion dna carrier
Make up
With embodiment 2 resulting fusion dnas is that MWPsp-MWPmp20-joint-Cleavage 1-GH22kd is a template, forward primer with 5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering 5), reverse primer with 5 '-AAACTCCTGGTAGGTGTCAAA-3 ' (sequence numbering 16), amplification of DNA fragments MWPsp-MWPmp20-joint-Cleavage1-GH (A), also use the forward primer of 5 '-AACCCCCAGACCTCCCTCTGT-3 ' (sequence numbering 17), with the reverse primer of 5 '-CTAGAAGCCACAGCTGCCCT-3 ' (sequence numbering 11), amplification of DNA fragments GH (B).By the ligation and the PCR reaction of combination embodiment 1 record, obtained inserting the carrier of fusion dna MWPsp-MWPmp20-joint-Cleavage 1-GH20kd by these two dna fragmentations then.
Inserted MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd fusion dna
The structure of carrier
With the method identical with embodiment 1 (2), the forward oligonucleotide of 5 '-CATCATCATCATCATCACGACTATGATATCCCGACCACTGAAAACCTGTACTTCCA AGGT (sequence numbering 19) of chemical synthesis coding aminoacid sequence His-His-His-His-His-His-Asp-Tyr-Asp-Ile-Pro-Thr-Thr-Glu-Asn-Leu-Tyr-Phe-Gln-Gly (sequence numbering 18), the reverse oligonucleotide of 5 '-ACCTTGGAAGTACAGGTTTTCAGTGGTCGGGATATCATAGTCGTGATGATGATGAT GATG-3 ' (sequence numbering 20) obtains dna fragmentation (His) 6-joint-Cleavage 2.Ligation by combination embodiment 1 record then, the dna fragmentation MWPsp-MWPmp20 that obtains by embodiment 1 and (His) 6-joint-Cleavage 2 obtain fusion dna MWPsp-MWPmp20-(His) 6-joint-Cleavage 2.
The dna fragmentation GH22kd that adopting uses the same method is again obtained by fusion dna that obtains like this and embodiment 1 (3) obtains to have inserted the carrier of fusion dna MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd.
Inserted the carrier of MWPsp-MWPmp20-joint-Cleavage 2-GH22kd fusion dna
Structure
The fusion dna MWPsp-MWPmp20-joint-Cleavage2-GH22kd that obtains with embodiment 6 is a template, forward primer with 5 '-GACTATGATATCCCGACCACT-3 ' (sequence numbering 14), with the reverse primer of 5 '-CTA GAAGCCACAGCTGCCCT-3 ' (sequence numbering 11), amplification of DNA fragments joint-Cleavage 2-GH22kd.Ligation thing PCR reaction by combination embodiment 1 record has then been obtained inserting the carrier of fusion dna MWPsp-MWPmp20-joint-Cleavage 2-GH22kd by the dna fragmentation of the dna fragmentation MWPsp-MWPmp20 of embodiment 1 (1) gained and joint-Cleavage 2-GH22kd.
Inserted the structure of the carrier of fusion dna MWPsp-GH22kd
Genomic dna 840ng with bacillus pumilus (Bacillus brevis 47-5Q bacterial strain) is a template, forward primer with 5 '-GTCGTTAACAGTGTATTGCT-3 ' (sequence numbering 5), with the reverse primer of 5 '-TGCGAAAGCCATTGGAGCAAC-3 ' (sequence numbering 21), amplification of DNA fragments MWPsp.Ligation and PCR by combination embodiment 1 record reacts then, by dna fragmentation MWPsp and the carrier that has been obtained inserting fusion dna MWPsp-GH22kd by the dna fragmentation GH22kd of embodiment 1 (3) gained.
The expression-secretion of fusion rotein and parsing
(1) amino acid of fusions and base sequence
The amino acid and the base sequence of embodiment 1～8 resulting fusions give numbering shown below respectively.
MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd (sequence numbering 22,30)
MWPsp-MWPmp20-joint-Cleavage 1-GH22kd (sequence numbering 23,31)
MWPsp-MWPmp20-Cleavage 1-GH22kd (sequence numbering 24,32)
MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd (sequence numbering 25,33)
MWPsp-MWPmp20-joint-Cleavage 1-GH20kd (sequence numbering 26,34)
MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd (sequence numbering 27,35)
MWPsp-MWPmp20-joint-Cleavage 2-GH22kd (sequence numbering 28,36)
MWPsp-GH22kd (sequence numbering 29,37)
(2) expression-secretion of fusions
Express their coded fused proteins with the various fusion dnas that embodiment 1～8 obtains.Its representational embodiment is that MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd is inserted into the mode in the expression vector as shown in Figure 1.
This method carrier of various fusion dnas that has been exactly insertion that embodiment 1～8 is obtained, with restriction enzyme A paL I and Hind III (when DNA is inserted by forward with the M13 primer with respect to definite fusion base sequence) or ApaL I and kpn I (when being reversed insertion) processing with respect to the M13 primer, carry out 0.8% sepharose then, cut out the dna fragmentation that contains various fusion dnas.Contain dna fragmentation that handlebar cuts out and sequence numbering and be the promotor of 40 MWP, and the bacillus pumilus expression vector pNU211R2L5 of SD sequence (putting down into communique 5 years-No. 304962 referring to Japanese publication) is with ApaL I and Hind III when fusion dna (oppositely insert then be Kpn I) cutting, the product of respectively getting after an amount of cutting is mixed, the dna ligation kit of producing product with precious wine reacted 30 minutes at 16 ℃, thereby fusion dna is inserted on the expression vector.Use resulting expression vector, by known method [Method inEnzymol., 217,23 (1993)] transform bacillus pumilus (Bacillus brevis 47-5Q bacterial strain (FERM P-7224: clear and 60 years-No. 58074 communiques of Japanese publication, publication is clear and 62 years-No. 201589 communiques), be seeded in T2 nutrient agar (T2 nutrient agar: polyprotein peptone 1%, meat extract 0.5%, yeast extract paste 0.2%, uridylic 0.1mg/ml, glucose 1%, erythromycin 10 μ g/ml, agar 1.5%, pH7) the middle transformant that obtains.
Transformant 37 ℃ of cultivations T2 substratum (removing the substratum of agar from the T2 nutrient agar) were used known method [Molecular Cloning 2 after 1 day
NdEd., A LaboratoryManual, Cold Spring Harbor Laboratory (1989)] purifying DNA plasmid, handle with ApaL I and Hind III (or Kpn I), confirm that each fusion dna inserts.For the transformant of confirming to have inserted fusion dna, the test expression-secretion is by the fused protein of the fusion dna coding that inserts.
Method is that handle uses the T2 substratum at 37 ℃ of bacteria suspensions of cultivating 1 day, and the ratio with 1/1000 adds liquid nutrient medium (polyprotein peptone 3%, yeast extract paste 0.4%, glucose 3%, MgSO
47H
2O 0.01%, MnSO
44H
2O 0.001%, erythromycin 10 μ g/ml, pH8) in, be divided in test tube (2ml/20ml test tube) or the triangular flask (50ml/500ml triangular flask) 30 ℃ and cultivated 4 days.
After cultivate finishing, nutrient solution obtained culture supernatant in centrifugal 2 minutes through 15000rpm, press known method (Laemmli, U.K., Nature, 227, pp.680-685) with the electrophoretic method analysing protein.Specific practice is: add 1 μ l damping fluid 1[125mMTris-HCl (pH6.8) in 10 μ l culture supernatant, 20% glycerine, 4%SDS, 10%2-mercaptoethanol], boiled 5 minutes, add 2 μ l damping fluid 2[250mM Tris-HCl (pH6.5) again, 50% glycerine, 0.5%BPB], with commercially available 15/25%SDS polyacrylamide gel carry out (First Chemical Corp.'s system) electrophoresis (electrophoretic buffer: 100mM Tris, 100mM Tricine, 0.1%SDS), use coomassie brilliant blue staining behind the electrophoresis, detection has or not expression-secretion.
Fig. 2 represents is to make that various syzygys are expressed, the proteinic SDS-PAGE image of excretory in substratum.Human growth hormone gene is close to the SDS-PAGE image (the 3rd swimming lane) of resulting MWPsp-GH22kd is identical with the pNU211R2L5 that does not contain foreign protein genes fully (the 2nd swimming lane) behind the MWP signal peptide, but directly dispose the aminoacid sequence of Leader, (His) 6, joint and Cleavage in the MWPsp back and dispose the syzygy of human growth hormone gene, the remarkable expression-secretion of fusion rotein (arrow) is found (the 4th～10 swimming lane).For the 22kd human growth hormone, shown in MWPsp-MWPmp-Cleavage 1-GH22kd (the 4th swimming lane), MWPsp-MWPmp20-joint-Cleavage 1-GH22kd (the 5th swimming lane), MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd (the 6th swimming lane), by fitting joint before Cleavage 1-GH22kd sequence, then the fused protein secretory volume obviously increases.In addition, by assembling (His) 6 sequences before joint-Cleavage 1-GH22kd, further increased secretory volume.The effect of configuration (His) 6 also can be found out from MWPsp-MWPmp20-joint-Cleavage 2-GH22kd (the 9th swimming lane), MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd (the 10th swimming lane).On the other hand, in the MWPsp-MWPmp20-joint-Cleavage l-GH20kd (the 7th swimming lane) of the human growth hormone of 20kd and MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd (the 8th swimming lane), also significantly increase the effect of secretory volume as can be seen by configuration (His) 6.
(3) Immunological Identification of human growth hormone
Protein immunoblotting method with one of immunological method is identified human growth hormone.Resulting various transformants processes in embodiment 9 (2) are cultivated, the nutrient solution that obtains obtained culture supernatant in centrifugal 2 minutes through 15000rpm, get this supernatant liquor 15 μ l by with embodiment 9 (2) used identical method electrophoresis after, with known method [Towbin, H. waiting people 76,4350-4354 (1979)] electricity consumption is imprinted on the nitrocellulose filter.Then film is immersed in and is dissolved in damping fluid 3 (20mM Tris-HCl (pH7.4), 150mM NaCl, in 5% the skim-milk solution that 0.1%Tween20) is made into 15 minutes, being placed on then with vibration immersion in rabbit anti-HGH (the Biostride company products) liquid of 2000 times of damping fluid 3 dilutions 30 minutes.Then, clean each 10 minutes 3 times with damping fluid 3 vibrations.Be immersed in again with vibration in the anti-rabbit igg antibody through the hydrogen peroxide enzyme labelling (the E-Y Laboratories company product) liquid of 2000 times of damping fluid 3 dilutions 30 minutes.
After finishing immersion, vibrate with damping fluid 3 and to clean 3 times, each 10 minutes, whether the ECL detection kit scrutineer tethelin of producing with ア マ シ ヤ system company existed.Method is pressed process specifications.As shown in Figure 3, all syzygys except that the pNU211R2L5 that does not insert the GH gene order fully (first swimming lane), be MWPsp-GH22kd (the 2nd swimming lane), MWPsp-MWPmp20-Cleavage 1-GH22kd (the 3rd swimming lane), MWPsp-MWPmp20-joint-Cleavage 1-GH22kd (the 4th swimming lane), MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd (the 5th swimming lane), MWPsp-MWPmp20-joint-Cleavage 1-GH20kd (the 6th swimming lane), MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd (the 7th swimming lane), MWPsp-MWPmp20-joint-Cleavage 2-GH22kd (the 8th swimming lane), among MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd (the 9th swimming lane), all there is the clear signal (arrow indication) with the fusion rotein of anti-HGH's antibody response to detect.As can be seen, it is dense that the colour developing band of arrow is pressed the order of MWPsp-MWPmp20-Cleavage 1-GH22kd (the 3rd swimming lane), MWPsp-MWPmp20-joint-Cleavage 1-GH22kd (the 4th swimming lane), MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd (the 5th swimming lane) 3, and secretory volume increases.That is to say, joint separately or joint the effect of increase secretory volume is arranged during with (His) 6 coexistences.Even for the syzygy of cutting off mode Cleavage 2, when joint separately or joint with (His) 6 exist simultaneously under the situation of (the 9th swimming lane), the effect of increase secretory volume is more arranged when existing than joint independent (the 8th swimming lane).In addition, the secretory volume of the syzygy of GH20kd lacking than GH22kd.And joint is with the DeGrain of (His) 6.On the other hand, for the MWPsp-GH22kd that the signal peptide back at MWP directly connects the human growth hormone group, weak output signal illustrates that secretory volume is considerably less.
(4) use the quantitatively expression amount of human growth hormone of high performance liquid chromatography (HPLC)
For the syzygy of using Cleavage 1, be MWPsp-MWPmp20-Cleavage1-GH22kd, MWPsp-MWPmp20-joint-Cleavage 1-GH22kd and MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd, by using human growth hormone in the HpLC quantitative culture medium, and simultaneously with negative control pNU211R2L5 with behind the MWP signal peptide gene, directly be connected the MWPsp-GH22kd that the GH22kd gene forms and compare, investigated Leader in MWPmp20, joint and (His) effect of the secretory volume in 6.
Cultivation is resulting various transformants in embodiment 9 (2), and the nutrient solution that obtains obtained supernatant liquor 194 μ l in centrifugal 20 minutes through 15000rpm, add 6 μ l 6N HCl and mix, through 15000rpm centrifugal 10 minutes again.Supernatant liquor is applied to the C4 post (Waters Symmetry300 4.6 * 250mm, 5 μ m) that process 0.1%TFA/45% acetonitrile balance is crossed, with 0.1%TFA/45-73% acetonitrile wash-out with the membrane filtration of 0.22 μ m.When with 2%/minute ratio when improving acetonitrile concentration, a peak appearred near 20.35 minutes, and quantitative this peak.Fig. 4 represents is the quantitative values of excretory tethelin in each clone's every liter of the substratum.Find only to have among the MWPsp-22kd (B post) secretion of pettiness, if but between MWP signal peptide MWPsp and GH22kd, insert Leader (MWPmp20)-Cleavage 1 (C post), Leader (MWPmp20)-joint-Cleavage 1 (D post), or Leader (MWPmp20)-(His) 6-joint-Cleavage 1 (E post), then secretory volume increases.
Embodiment 10
From fused protein cutting and analyst's tethelin
The transformant that will contain the expression vector that has inserted MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd, MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd and MWPsp-MWPmp20-(His) 6-joint-fusion dnas such as Cleavage 2-GH22kd is cultivated a night, the gained nutrient solution is with 1/1000 ratio, be inoculated into (totally 10) in the 500ml triangular flask that the 50ml substratum is housed, cultivated 4 days for 30 ℃, wherein used identical in the expression among medium component and the embodiment 9 (2).The gained nutrient solution 4 ℃ with 9500rpm centrifugal 20 minutes.In the culture supernatant that obtains, add ultimate density and be 40% ammonium sulfate, place more than 1 hour at 4 ℃, again 4 ℃ with 9500rpm centrifugal 20 minutes.The dissolved in distilled water that in throw out, adds 1/2 volume, centrifugal 20 minutes again with 20000rpm.Sample on the supernatant liquor in 20mM phosphoric acid buffer (pH8) equilibrated nickel chelate column (ChelatingSepharose HP, Amersham Pharmacia Biotech) and be adsorbed onto on the resin, with the imidazoles wash-out of 90mM, is obtained containing the fraction of fusion rotein.In this fraction, add urea and Virahol then respectively, make its concentration separately reach 1M and 20%, place more than 1 hour at 4 ℃.Then to 20mMTris HCl damping fluid (pH8)/1mM EDTA dialysis, last sample is to anion-exchange column (the Q SepharoseHP that crosses through the same buffer balance, Amersham Pharmacia Biotech) goes up and be adsorbed onto on the resin, contain the fusion rotein of required product tethelin with the NaCl eluant solution of 0.05～0.3M.
In order to cut out tethelin, carry out following enzyme and handle from fusion rotein.To having the fusion rotein that Cleavage 1 is the aminoacid sequence of Ile-Glu-Gly-Arg (sequence numbering 1), be MWPmp20-(His) 6-joint-Cleavage 1-GH22kd, MWPmp20-(His) 6-joint-Cleavage 1-GH20kd, at 50mMTris HCl damping fluid (pH8)/100mMNaCl/5mM CaCl
2In, per 100 μ g fusion roteins added the factor Xa (Novagen) of 1 unit, in 8 ℃ of reactions 6 days.Can cut out the human growth hormone GH22kd and the GH20kd of natural type like this from fused protein.
To having Cleavage 2 is the Glu-Asn-Leu-Tyr-Phe-Gln-Gly (fusion rotein of the aminoacid sequence of (sequence numbering 2), be MWPmp20-(His) 6-joint-Cleavage 2-GH22kd, in 20mMTris HCl damping fluid (pH8)/1mM EDTA/10Mm DTT, add 1 TEV of unit proteolytic enzyme (Invitrogen) with respect to per 50 μ g fusion roteins, in room temperature (25 ℃) reaction 1 (16 hours) round the clock.Can cut out the human growth hormone that N-terminal has glycine residue from fusion rotein like this.
For the human growth hormone of from above-mentioned reaction product, purifying, sample on the above-mentioned reactant is gone up and made its absorption, this reversed-phase column use 0.1%TFA/10% acetonitrile balance mistake in advance to reversed-phase column (Resource RPC, Amersham Pharmacia Biotech).Acetonitrile wash-out with 38-52%.To the dialysis of 20mMTris HCl damping fluid, can obtain needed tethelin again.
The human growth hormone of selling in market (Norditropin), and the natural type human growth hormone 22kd and the 20kd of separation and purification, and to add ultimate density among the residual anomaly tethelin 22kd that glycine residue arranged of N-terminal be 0.5% TFA, go up sample again at C4 post (the Waters Symmetry300 that crosses with 0.1%TFA/40% acetonitrile balance in advance, 4.6 * 250mm, 5 μ m) on, use 0.1TFA/52-55% acetonitrile wash-out again.Elution curve as shown in Figure 5.
Natural type human growth hormone 22kd (B), N-terminal be residual the identical of the elution peak figure of anomaly tethelin 22kd (C) of glycine residue and commercially available tethelin (A), but natural type human growth hormone 20kd (D) then elution peak occurs on different positions, this has reflected the difference of molecular weight.
Embodiment 11
The bioactive mensuration of natural type human growth hormone 22kd, 20kd and anomaly tethelin
The fraction that will contain the tethelin peak that goes out by the HPLC wash-out among the embodiment 10 is done admittedly, is dissolved in the distilled water, gets its part and use the HPLC wash-out under above-mentioned the same terms, tries to achieve concentration according to resulting peak area.This moment, the peak area with commercially available tethelin was a standard, had adjusted the concentration of each tethelin with 0.25%BSA/0.9NaCl% solution.Carry out following biological activity test with them.The biological activity of human growth hormone is studied the influence of growth by use the back in the rat of excision pituitary body.Every 35 age in days female Wistar of extracing behind the pituitary body the 4th day are rat, the per 4 days subcutaneous testing samples of giving with 0.05 unit, 7 every group.As negative control, use 0.25%BSA/0.9%NaCl solution.The dispensing back was killed rat on the 4th day, and it is long to take out Thigh bone measurement bone.Measured the body weight during offeing medicine in the lump.Detect significant difference with the Xue Shengshi t method of inspection.Fig. 6 has represented the situation that dispensing back body weight changes.Negative control is not observed weight increase, and determined sample, comprises that N-terminal has that anomaly GH22kd, the natural type GH22kd of glycine residue is the same with positive control with natural type GH20kd an effect of putting on weight.
In the long measurement of Thigh bone bone, also see the animal of throwing determined sample, the same effect (Fig. 7) that increase bone length is arranged with positive control.
The industry practicality
As mentioned above, rely on the present invention, set up and increased considerably gene recombinant human growth hormone Output, a large amount of produce active GH22kd and functional fragment thereof are arranged, variant Or the technology of similar thing.
Sequence table
＜110〉ITOHAM FOODS INC (ITOHAM FOODS INC.)
＜120〉be used for the DNA and the application thereof of tethelin high expression level
(DNA?for?high?expression?of?growth?hormone，and?use?thereof)
<130>SCT034073-47
<160>43
<210>1
<211>4
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Cleavage 1 generates GH as the cleavage site of fusion rotein and factor Xa
<400>1
Ile?Glu?Gly?Arg
1
<210>2
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉Cleavage 2 generates GH as the cleavage site of fusion rotein and TEV proteolytic enzyme
<400>2
Glu?Asn?Leu?Tyr?Phe?Gln?Gly
1 5
<210>3
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉effectively produce candidate's joint of GH
<400>3
Asp?Tyr?Asp?Ile?Pro?Thr?Thr
1 5
<210>4
<211>7
<212>PRT
＜213〉artificial sequence
<220>
＜223〉candidate Cleavage generates GH as the cleavage site of fusion rotein and TEV proteolytic enzyme
<400>4
Glu?Asn?Leu?Tyr?Phe?Gln?Ser
1 5
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for containing the forward primer of the DNA of MWPsp-MWPmp20 at its 5 ' end pcr amplification
<400>5
gtcgttaaca?gtgtattgct 20
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the reverse primer of pcr amplification MWPsp-MWPmp20 DNA
<400>6
tacggttttt?tccatatcag?c 21
<210>7
<211>17
<212>PRT
＜213〉artificial sequence
<220>
＜223〉contain the designed peptide of joint and Cleavage, (His) 6-joint-Cleavage 1
<400>7
His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr?Thr?Ile?Glu?Gly
1 5 10 15
Arg
<210>8
<211>51
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the forward oligonucleotide of coding (His) 6-joint-Cleavage 1
<400>8
catcatcatc?atcatcacga?ctatgatatc?ccgaccacta?tcgaaggtcg?t 51
<210>9
<211>51
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse oligonucleotide of coding (His) 6-joint-Cleavage 1
<400>9
acgaccttcg?atagtggtcg?ggatatcata?gtcgtgatga?tgatgatgat?g 51
<210>10
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the forward primer of the DNA of pcr amplification coding GH22kd
<400>10
atggctacag?gctcccggac 20
<210>11
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for comprising the reverse primer of DNA of the sequence of the GH22kd that encodes at its 3 ' end pcr amplification
<400>11
ctagaagcca?cagctgccct 20
<210>12
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the forward primer of pcr amplification GH22kd
<400>12
ttcccaacca?ttcccttatc 20
<210>13
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the reverse primer of the fusion dna of pcr amplification called after MWPsp-MWPmp20-(His) 6-joint-Cleavage 1
<400>13
acgaccttcg?atagtggtcg?g 21
<210>14
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the forward primer that the pcr amplification fusion dna is joint-Cleavage 1-GH22kd
<400>14
gactatgata?tcccgaccac?t 21
<210>15
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the forward primer that the pcr amplification fusion dna is Cleavage 1-GH22kd
<400>15
atcgaaggtc?gtttcccaac?c 21
<210>16
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the reverse primer of the dna fragmentation of pcr amplification called after MWPsp-MWPmp20-joint-Cleavage 1-GH (A)
<400>16
aaactcctgg?taggtgtcaa?a 21
<210>17
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the forward primer of the DNA of pcr amplification coding human growth hormone fragment GH (B)
<400>17
aacccccaga?cctccctctg?t 21
<210>18
<211>20
<212>PRT
＜213〉artificial sequence
<220>
＜223〉She Ji fusion rotein, (His) 6-joint-Cleavage 2
<400>18
His?His?His?His?His?His?Asp?Tyr?Asp?Ile?Pro?Thr?Thr?Glu?Asn?Leu
1 5 10 15
Tyr?Phe?Gln?Gly
20
<210>19
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the forward oligonucleotide of coding (His) 6-joint-Cleavage 2
<400>19
catcatcatc?atcatcacga?ctatgatatc?ccgaccactg?aaaacctgta?cttccaaggt 60
<210>20
<211>60
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse oligonucleotide of coding (His) 6-joint-Cleavage 2
<400>20
accttggaag?tacaggtttt?cagtggtcgg?gatatcatag?tcgtgatgat?gatgatgatg 60
<210>21
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the reverse primer of pcr amplification MWPsp
<400>21
tgcgaaagcc?attggagcaa?c 21
<210>22
<211>248
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd
<400>22
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?His?His?His?His?His?His?Asp?Tyr
35 40 45
Asp?Ile?Pro?Thr?Thr?Ile?Glu?Gly?Arg?Phe?Pro?Thr?Ile?Pro?Leu?Ser
50 55 60
Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu
65 70 75 80
Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys?Glu
85 90 95
Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser
100 105 110
Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser
115 120 125
Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu
130 135 140
Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr
145 150 155 160
Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu
165 170 175
Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr
180 185 190
Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser?His
195 200 205
Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg
210 215 220
Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg
225 230 235 240
Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
245
<210>23
<211>242
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-joint-Cleavage 1-GH22kd
<400>23
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?Asp?Tyr?Asp?Ile?Pro?Thr?Thr?Ile
35 40 45
Glu?Gly?Arg?Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala
50 55 60
Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln
65 70 75 80
Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu
85 90 95
Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro
100 105 110
Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg
115 120 125
Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu
130 135 140
Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn
145 150 155 160
Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met
165 170 175
Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln
180 185 190
Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu
195 200 205
Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val
210 215 220
Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys
225 230 235 240
Gly?Phe
<210>24
<211>235
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-Cleavage 1-GH22kd
<400>24
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?Ile?Glu?Gly?Arg?Phe?Pro?Thr?Ile
35 40 45
Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu
50 55 60
His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile
65 70 75 80
Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu
85 90 95
Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln
100 105 110
Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln
115 120 125
Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser
130 135 140
Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp
145 150 155 160
Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser
165 170 175
Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr
180 185 190
Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr
195 200 205
Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val
210 215 220
Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
225 230 235
<210>25
<211>233
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd
<400>25
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?His?His?His?His?His?His?Asp?Tyr
35 40 45
Asp?Ile?Pro?Thr?Thr?Ile?Glu?Gly?Arg?Phe?Pro?Thr?Ile?Pro?Leu?Ser
50 55 60
Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu
65 70 75 80
Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe
85 90 95
Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys
100 105 110
Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp
115 120 125
Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val
130 135 140
Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu
145 150 155 160
Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg
165 170 175
Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser
180 185 190
His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe
195 200 205
Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys
210 215 220
Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
225 230
<210>26
<211>227
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-joint-Cleavage 1-GH20kd
<400>26
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?Asp?Tyr?Asp?Ile?Pro?Thr?Thr?Ile
35 40 45
Glu?Gly?Arg?Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala
50 55 60
Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln
65 70 75 80
Glu?Phe?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr
85 90 95
Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu
100 105 110
Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe
115 120 125
Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser
130 135 140
Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu
145 150 155 160
Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys
165 170 175
Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu
180 185 190
Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys
195 200 205
Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser
210 215 220
Cys?Gly?Phe
225
<210>27
<211>251
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd
<400>27
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?His?His?His?His?His?His?Asp?Tyr
35 40 45
Asp?Ile?Pro?Thr?Thr?Glu?Asn?Leu?Tyr?Phe?Gln?Gly?Phe?Pro?Thr?Ile
50 55 60
Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu
65 70 75 80
His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile
85 90 95
Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu
100 105 110
Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln
115 120 125
Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln
130 135 140
Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser
145 150 155 160
Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp
165 170 175
Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser
180 185 190
Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr
195 200 205
Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr
210 215 220
Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val
225 230 235 240
Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
245 250
<210>28
<211>245
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-MWPmp20-joint-Cleavage 2-GH22kd
<400>28
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala?Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met
20 25 30
Asp?Ala?Asp?Met?Glu?Lys?Thr?Val?Asp?Tyr?Asp?Ile?Pro?Thr?Thr?Glu
35 40 45
Asn?Leu?Tyr?Phe?Gln?Gly?Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe
50 55 60
Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp
65 70 75 80
Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr
85 90 95
Ser?Phe?Leu?Gln?Ash?Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile
100 105 110
Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu
115 120 125
Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val
130 135 140
Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser
145 150 155 160
Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln
165 170 175
Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile
180 185 190
Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp
195 200 205
Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met
210 215 220
Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu
225 230 235 240
Gly?Ser?Cys?Gly?Phe
245
<210>29
<211>211
<212>PRT
＜213〉artificial sequence
<220>
＜223〉fusion rotein called after MWPsp-GH22kd
<400>29
Val?Val?Asn?Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro
1 5 10 15
Met?Ala?Phe?Ala?Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn
20 25 30
Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr
35 40 45
Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe
50 55 60
Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr
65 70 75 80
Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu
85 90 95
Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe
100 105 110
Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser
115 120 125
Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu
130 135 140
Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys
145 150 155 160
Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu
165 170 175
Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys
180 185 190
Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser
195 200 205
Cys?Gly?Phe
210
<210>30
<211>744
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH22kd
<400>30
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
catcatcatc?atcatcacga?ctatgatatc?ccgaccacta?tcgaaggtcg?tttcccaacc 180
attcccttat?ccaggctttt?tgacaacgct?atgctccgcg?cccatcgtct?gcaccagctg 240
gcctttgaca?cctaccagga?gtttgaagaa?gcctatatcc?caaaggaaca?gaagtattca 300
ttcctgcaga?acccccagac?ctccctctgt?ttctcagagt?ctattccgac?accctccaac 360
agggaggaaa?cacaacagaa?atccaaccta?gagctgctcc?gcatctccct?gctgctcatc 420
cagtcgtggc?tggagcccgt?gcagttcctc?aggagtgtct?tcgccaacag?cctggtgtac 480
ggcgcctctg?acagcaacgt?ctatgacctc?ctaaaggacc?tagaggaagg?catccaaacg 540
ctgatgggga?ggctggaaga?tggcagcccc?cggactgggc?agatcttcaa?gcagacctac 600
agcaagttcg?acacaaactc?acacaacgat?gacgcactac?tcaagaacta?cgggctgctc 660
tactgcttca?ggaaggacat?ggacaaggtc?gagacattcc?tgcgcatcgt?gcagtgccgc 720
tctgtggagg?gcagctgtgg?cttc 744
<210>31
<211>726
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-joint-Cleavage 1-GH22kd
<400>31
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
gactatgata?tcccgaccac?tatcgaaggt?cgtttcccaa?ccattccctt?atccaggctt 180
tttgacaacg?ctatgctccg?cgcccatcgt?ctgcaccagc?tggcctttga?cacctaccag 240
gagtttgaag?aagcctatat?cccaaaggaa?cagaagtatt?cattcctgca?gaacccccag 300
acctccctct?gtttctcaga?gtctattccg?acaccctcca?acagggagga?aacacaacag 360
aaatccaacc?tagagctgct?ccgcatctcc?ctgctgctca?tccagtcgtg?gctggagccc 420
gtgcagttcc?tcaggagtgt?cttcgccaac?agcctggtgt?acggcgcctc?tgacagcaac 480
gtctatgacc?tcctaaagga?cctagaggaa?ggcatccaaa?cgctgatggg?gaggctggaa 540
gatggcagcc?cccggactgg?gcagatcttc?aagcagacct?acagcaagtt?cgacacaaac 600
tcacacaacg?atgacgcact?actcaagaac?tacgggctgc?tctactgctt?caggaaggac 660
atggacaagg?tcgagacatt?cctgcgcatc?gtgcagtgcc?gctctgtgga?gggcagctgt 720
ggcttc 726
<210>32
<211>705
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-Cleavage 1-GH22kd
<400>32
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
atcgaaggtc?gtttcccaac?cattccctta?tccaggcttt?ttgacaacgc?tatgctccgc 180
gcccatcgtc?tgcaccagct?ggcctttgac?acctaccagg?agtttgaaga?agcctatatc 240
ccaaaggaac?agaagtattc?attcctgcag?aacccccaga?cctccctctg?tttctcagag 300
tctattccga?caccctccaa?cagggaggaa?acacaacaga?aatccaacct?agagctgctc 360
cgcatctccc?tgctgctcat?ccagtcgtgg?ctggagcccg?tgcagttcct?caggagtgtc 420
ttcgccaaca?gcctggtgta?cggcgcctct?gacagcaacg?tctatgacct?cctaaaggac 480
ctagaggaag?gcatccaaac?gctgatgggg?aggctggaag?atggcagccc?ccggactggg 540
cagatcttca?agcagaccta?cagcaagttc?gacacaaact?cacacaacga?tgacgcacta 600
ctcaagaact?acgggctgct?ctactgcttc?aggaaggaca?tggacaaggt?cgagacattc 660
ctgcgcatcg?tgcagtgccg?ctctgtggag?ggcagctgtg?gcttc 705
<210>33
<211>699
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-(His) 6-joint-Cleavage 1-GH20kd
<400>33
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
catcatcatc?atcatcacga?ctatgatatc?ccgaccacta?tcgaaggtcg?tttcccaacc 180
attcccttat?ccaggctttt?tgacaacgct?atgctccgcg?cccatcgtct?gcaccagctg 240
gcctttgaca?cctaccagga?gtttaacccc?cagacctccc?tctgtttctc?agagtctatt 300
ccgacaccct?ccaacaggga?ggaaacacaa?cagaaatcca?acctagagct?gctccgcatc 360
tccctgctgc?tcatccagtc?gtggctggag?cccgtgcagt?tcctcaggag?tgtcttcgcc 420
aacagcctgg?tgtacggcgc?ctctgacagc?aacgtctatg?acctcctaaa?ggacctagag 480
gaaggcatcc?aaacgctgat?ggggaggctg?gaagatggca?gcccccggac?tgggcagatc 540
ttcaagcaga?cctacagcaa?gttcgacaca?aactcacaca?acgatgacgc?actactcaag 600
aactacgggc?tgctctactg?cttcaggaag?gacatggaca?aggtcgagac?attcctgcgc 660
atcgtgcagt?gccgctctgt?ggagggcagc?tgtggcttc 699
<210>34
<211>681
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-joint-Cleavage 1-GH20kd
<400>34
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
gactatgata?tcccgaccac?tatcgaaggt?cgtttcccaa?ccattccctt?atccaggctt 180
tttgacaacg?ctatgctccg?cgcccatcgt?ctgcaccagc?tggcctttga?cacctaccag 240
gagtttaacc?cccagacctc?cctctgtttc?tcagagtcta?ttccgacacc?ctccaacagg 300
gaggaaacac?aacagaaatc?caacctagag?ctgctccgca?tctccctgct?gctcatccag 360
tcgtggctgg?agcccgtgca?gttcctcagg?agtgtcttcg?ccaacagcct?ggtgtacggc 420
gcctctgaca?gcaacgtcta?tgacctccta?aaggacctag?aggaaggcat?ccaaacgctg 480
atggggaggc?tggaagatgg?cagcccccgg?actgggcaga?tcttcaagca?gacctacagc 540
aagttcgaca?caaactcaca?caacgatgac?gcactactca?agaactacgg?gctgctctac 600
tgcttcagga?aggacatgga?caaggtcgag?acattcctgc?gcatcgtgca?gtgccgctct 660
gtggagggca?gctgtggctt?c 681
<210>35
<211>753
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-(His) 6-joint-Cleavage 2-GH22kd
<400>35
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
catcatcatc?atcatcacga?ctatgatatc?ccgaccactg?aaaacctgta?cttccaaggt 180
ttcccaacca?ttcccttatc?caggcttttt?gacaacgcta?tgctccgcgc?ccatcgtctg 240
caccagctgg?cctttgacac?ctaccaggag?tttgaagaag?cctatatccc?aaaggaacag 300
aagtattcat?tcctgcagaa?cccccagacc?tccctctgtt?tctcagagtc?tattccgaca 360
ccctccaaca?gggaggaaac?acaacagaaa?tccaacctag?agctgctccg?catctccctg 420
ctgctcatcc?agtcgtggct?ggagcccgtg?cagttcctca?ggagtgtctt?cgccaacagc 480
ctggtgtacg?gcgcctctga?cagcaacgtc?tatgacctcc?taaaggacct?agaggaaggc 540
atccaaacgc?tgatggggag?gctggaagat?ggcagccccc?ggactgggca?gatcttcaag 600
cagacctaca?gcaagttcga?cacaaactca?cacaacgatg?acgcactact?caagaactac 660
gggctgctct?actgcttcag?gaaggacatg?gacaaggtcg?agacattcct?gcgcatcgtg 720
cagtgccgct?ctgtggaggg?cagctgtggc?ttc 753
<210>36
<211>735
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-MWPmp20-joint-Cleavage 2-GH22kd
<400>36
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
gcagaagaag?cagcaactac?tacagctcca?aaaatggacg?ctgatatgga?aaaaaccgta 120
gactatgata?tcccgaccac?tgaaaacctg?tacttccaag?gtttcccaac?cattccctta 180
tccaggcttt?ttgacaacgc?tatgctccgc?gcccatcgtc?tgcaccagct?ggcctttgac 240
acctaccagg?agtttgaaga?agcctatatc?ccaaaggaac?agaagtattc?attcctgcag 300
aacccccaga?cctccctctg?tttctcagag?tctattccga?caccctccaa?cagggaggaa 360
acacaacaga?aatccaacct?agagctgctc?cgcatctccc?tgctgctcat?ccagtcgtgg 420
ctggagcccg?tgcagttcct?caggagtgtc?ttcgccaaca?gcctggtgta?cggcgcctct 480
gacagcaacg?tctatgacct?cctaaaggac?ctagaggaag?gcatccaaac?gctgatgggg 540
aggctggaag?atggcagccc?ccggactggg?cagatcttca?agcagaccta?cagcaagttc 600
gacacaaact?cacacaacga?tgacgcacta?ctcaagaact?acgggctgct?ctactgcttc 660
aggaaggaca?tggacaaggt?cgagacattc?ctgcgcatcg?tgcagtgccg?ctctgtggag 720
ggcagctgtg?gcttc 735
<210>37
<211>633
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the design of coding MWPsp-GH22kd
<400>37
gtcgttaaca?gtgtattggc?tagtgcactc?gcacttactg?ttgctccaat?ggctttcgca 60
ttcccaacca?ttcccttatc?caggcttttt?gacaacgcta?tgctccgcgc?ccatcgtctg 120
caccagctgg?cctttgacac?ctaccaggag?tttgaagaag?cctatatccc?aaaggaacag 180
aagtattcat?tcctgcagaa?cccccagacc?tccctctgtt?tctcagagtc?tattccgaca 240
ccctccaaca?gggaggaaac?acaacagaaa?tccaacctag?agctgctccg?catctccctg 300
ctgctcatcc?agtcgtggct?ggagcccgtg?cagttcctca?ggagtgtctt?cgccaacagc 360
ctggtgtacg?gcgcctctga?cagcaacgtc?tatgacctcc?taaaggacct?agaggaaggc 420
atccaaacgc?tgatggggag?gctggaagat?ggcagccccc?ggactgggca?gatcttcaag 480
cagacctaca?gcaagttcga?cacaaactca?cacaacgatg?acgcactact?caagaactac 540
gggctgctct?actgcttcag?gaaggacatg?gacaaggtcg?agacattcct?gcgcatcgtg 600
cagtgccgct?ctgtggaggg?cagctgtggc?ttc 633
<210>38
<211>217
<212>PRT
＜213〉homo sapiens
<300>
＜303〉genome
<304>4
<306>479-497
<307>1989
<400>38
Met?Ala?Thr?Gly?Ser?Arg?Thr?Ser?Leu?Leu?Leu?Ala?Phe?Gly?Leu?Leu
1 5 10 15
Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala?Phe?Pro?Thr?Ile?Pro?Leu
20 25 30
Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln
35 40 45
Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys
50 55 60
Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe
65 70 75 80
Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys
85 90 95
Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp
100 105 110
Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val
115 120 125
Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu
130 135 140
Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg
145 150 155 160
Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser
165 170 175
His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe
180 185 190
Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys
195 200 205
Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
210 215
<210>39
<211>217
<212>PRT
＜213〉homo sapiens
<300>
＜303〉genome
<304>4
<306>479-497
<307>1989
<400>39
Met?Ala?Ala?Gly?Ser?Arg?Thr?Ser?Leu?Leu?Leu?Ala?Phe?Gly?Leu?Leu
1 5 10 15
Cys?Leu?Ser?Trp?Leu?Gln?Glu?Gly?Ser?Ala?Phe?Pro?Thr?Ile?Pro?Leu
20 25 30
Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?Arg?Arg?Leu?Tyr?Gln
35 40 45
Leu?Ala?Tyr?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Leu?Lys
50 55 60
Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe
65 70 75 80
Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Val?Lys?Thr?Gln?Gln?Lys
85 90 95
Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Set?Leu?Leu?Leu?Ile?Gln?Ser?Trp
100 105 110
Leu?Glu?Pro?Val?Gln?Leu?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val
115 120 125
Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Arg?His?Leu?Lys?Asp?Leu?Glu
130 135 140
Glu?Gly?Ile?Gln?Thr?Leu?Met?Trp?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg
145 150 155 160
Thr?Gly?Gln?Ile?Phe?Asn?Gln?Ser?Tyr?Ser?Lys?Phe?Asp?Thr?Lys?Ser
165 170 175
His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe
180 185 190
Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys
195 200 205
Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
210 215
<210>40
<211>1200
<212>DNA
＜213〉bacillus pumilus (Bacillus brevis)
<220>
<221>CDS
<222>(621)...(＞1220)
<220>
＜221〉sig_ peptide
<222>(621)...(689)
<220>
＜221〉mat_ peptide
<222>(690)...(＞1217)
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 1 "
<222>260
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 2 "
<222>374
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 3 "
<222>444
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 4 "
<222>490
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 5 "
<222>504
<300>
<303>J.Bacteriol.
<304>169
<306>1239-1245
<307>1987
<400>40
cagctgagag?ctatcgcttg?aaaaatttgc?gttatgaaaa?tggacttgcg?acgactttag 60
aggtcattca?atcggaagaa?acattgtcta?ctcgtgagaa?tgcgtaccaa?aaagctatcc 120
tgtcttacaa?cttggctgtt?gtaaactttg?aaaatgcatt?aggaaattaa?cctaattcaa 180
gcaagattat?gaggttttga?accaaattgg?aaaaaggttc?agtcgtgaca?gcccgccata 240
tgtcccctat?aatacggatt?gtggcggatg?tcacttcgta?cataatggac?aggtgaataa 300
cgaaccacga?aaaaaacttt?aaattttttt?cgaaggcgcc?gcaacttttg?attcgctcag 360
gcgtttaata?ggatgtcaca?cgaaaaacgg?ggaattgtgt?aaaaaagatt?cacgaattct 420
agcagttgtg?ttacactagt?gattgttgca?ttttacacaa?tactgaatat?actagagatt 480
tttaacacaa?aaagcgaggc?tttcctgcga?aaggaggtga?cacgcgcttg?caggattcgg 540
gctttaaaaa?gaaagataga?ttaacaacaa?atattcccca?agaacaattt?gtttatacta 600
gaggaggaga?acacaaggtt?atg?aaa?aag?gtc?gtt?aac?agt?gta?ttg?gct?agt 653
Met?Lys?Lys?Val?Val?Asn?Ser?Val?Leu?Ala?Ser
1 5 10
gca?ctc?gca?ctt?act?gtt?gct?cca?atg?gct?ttc?gca?gca?gaa?gaa?gca 701
Ala?Leu?Ala?Leu?Thr?Val?Ala?Pro?Met?Ala?Phe?Ala?Ala?Glu?Glu?Ala
15 20 25
gca?act?act?aca?gct?cca?aaa?atg?gac?gct?gat?atg?gaa?aaa?acc?gta 749
Ala?Thr?Thr?Thr?Ala?Pro?Lys?Met?Asp?Ala?Asp?Met?Glu?Lys?Thr?Val
30 35 40
aaa?cgt?ctg?gaa?gct?ctt?ggc?ctg?gta?gca?ggt?tat?ggc?aac?ggc?gaa 797
Lys?Arg?Leu?Glu?Ala?Leu?Gly?Leu?Val?Ala?Gly?Tyr?Gly?Asn?Gly?Glu
45 50 55
tac?ggt?gta?gac?aaa?act?atc?act?cgt?gca?gag?ttc?gct?act?ctg?gtt 845
Tyr?Gly?Val?Asp?Lys?Thr?Ile?Thr?Arg?Ala?Glu?Phe?Ala?Thr?Leu?Val
60 65 70 75
gtt?cgc?gct?cgc?gga?ctg?gag?caa?ggt?gcg?aaa?ttg?gca?caa?ttc?agc 893
Val?Arg?Ala?Arg?Gly?Leu?Glu?Gln?Gly?Ala?Lys?Leu?Ala?Gln?Phe?Ser
80 85 90
aat?act?tac?aca?gat?gtg?aaa?tct?act?gat?tgg?ttt?gct?ggt?ttc?gta 941
Asn?Thr?Tyr?Thr?Asp?Val?Lys?Ser?Thr?Asp?Trp?Phe?Ala?Gly?Phe?Val
95 100 105
aac?gta?gct?tcc?ggc?gaa?gaa?atc?gta?aaa?ggt?ttc?ccg?gac?aaa?tct 989
Asn?Val?Ala?Ser?Gly?Glu?Glu?Ile?Val?Lys?Gly?Phe?Pro?Asp?Lys?Ser
110 115 120
ttc?aaa?cca?caa?aac?caa?gtt?act?tat?gct?gaa?gct?gta?act?atg?atc 1037
Phe?Lys?Pro?Gln?Asn?Gln?Val?Thr?Tyr?Ala?Glu?Ala?Val?Thr?Met?Ile
125 130 135
gtt?cgt?gca?ctg?ggt tat?gag?cca?tcc?gtt?aag?ggt?gta?tgg?cct?aac 1085
Val?Arg?Ala?Leu?Gly?Tyr?Glu?Pro?Ser?Val?Lys?Gly?Val?Trp?Pro?Asn
140 145 150 155
agc?atg?atc?tcc?aaa?gct?tcc?gag?ctg?aac?att?gct?aga?agc?atc?act 1133
Ser?Met?Ile?Ser?Lys?Ala?Ser?Glu?Leu?Asn?Ile?Ala?Arg?Ser?Ile?Thr
160 165 170
act?cct?aac?aat?gca?gca?act?cgt?ggc?gat?atc?ttc?aaa?atg?ctc?gac 1181
Thr?Pro?Asn?Asn?Ala?Ala?Thr?Arg?Gly?Asp?Ile?Phe?Lys?Met?Leu?Asp
175 180 185
aac?gct?ctt?cgc?gta?gac?ctg?atg?gag?caa?gtt?gaa?ttc 1220
Asn?Ala?Leu?Arg?Val?Asp?Leu?Met?Glu?Gln?Val?Glu?Phe
190 195 200
<210>41
<211>4330
<212>DNA
＜213〉bacillus pumilus (Bacillus brevis)
<220>
<221>CDS
<222>(548)...(3898)
<220>
＜221〉sig_ peptide
<222>(548)...(706)
<220>
＜221〉mat_ peptide
<222>(707)...(3895)
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 1 "
<222>280
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 2 "
<222>390
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 3 "
<222>460
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 4 "
<222>508
<220>
＜221〉misc_ feature
/ note=" transcription initiation site 5 "
<222>521
<300>
<303>J.Bacteriol.
<304>172
<306>1312-1320
<307>1990
<400>41
atcgattacc?aaaaaaccgc?gaaagattcc?gcggctgaga?gctatcgttt?gaccaatttg 60
cgctttgaaa?cggtctggca?acgaccctcg?aagtgattca?gccgaggagg?aattatccaa 120
ccgtgagaat?cagtaccaaa?aagcgatcct?gcagtacaat?ctggctgtgg?taaactttga 180
aactgccctc?ggaagctaag?aagaatttgt?aaggttttga?atggaactgg?aaaaaggttc 240
aatggtgaca?gtccgtccgg?cgtaccctat?aatacgagtt?gtggcggatg?tcactgctta 300
catattacaa?gtgaatacga?cccatgaaaa?tttctttaaa?ctttttttct?gaggcgccgc 360
aacttttgct?cgactgaggc?gtttaatagg?gtgtcacacg?aaaaacgggg?aattgtgtaa 420
aaaagattca?cggattctag?catttgtgtt?acactagtga?ttgttacatt?ttacacaata 480
gctgaatata?ctagagattt?ttaacacaaa?aaagcgaagc?tgtcctgcga?aaggaggtga 540
cacgcgcttg?caggattcgg?gctttaaaaa?gaaagataga?tcaacaaata?ttccccaaga 600
acaatttgtt?tatactagag?gaggagaaca?caaggtt?atg?aaa?aag?gtc?gtt?aac 655
Met?Lys?Lys?Val?Val?Asn
1 5
agt?gta?ttg?gct?agt?gcg?ctc?gcc?atc?aca?gtt?gct?cca?atg?gct?ttc 703
Ser?Val?Leu?Ala?Ser?Ala?Leu?Ala?Ile?Thr?Val?Ala?Pro?Met?Ala?Phe
10 15 20
gca?gca?gag?gac?aca?act?aca?gct?cct?aaa?atg?gat?gct?gca?atg?gag 751
Ala?Ala?Glu?Asp?Thr?Thr?Thr?Ala?Pro?Lys?Met?Asp?Ala?Ala?Met?Glu
25 30 35
aaa?acc?gta?aaa?cgt?ctg?gaa?gct?ctt?ggc?ctg?gta?gca?ggt?tat?ggc 799
Lys?Thr?Val?Lys?Arg?Leu?Glu?Ala?Leu?Gly?Leu?Val?Ala?Gly?Tyr?Gly
40 45 50
aac?ggc?gat?ttc?ggt?gca?gac?aaa?acc?atc?act?cgt?gca?gag?ttc?gct 847
Asn?Gly?Asp?Phe?Gly?Ala?Asp?Lys?Thr?Ile?Thr?Arg?Ala?Glu?Phe?Ala
55 60 65
act?cta?atc?gtt?cgc?gct?cgc?ggt?ctg?gag?caa?ggt?gcg?aaa?ctg?gca 895
Thr?Leu?Ile?Val?Arg?Ala?Arg?Gly?Leu?Glu?Gln?Gly?Ala?Lys?Leu?Ala
70 75 80 85
caa?ttc?aac?act?act?tac?aca?gat?gtt?aga?tcc?act?gac?tgg?ttc?gct 943
Gln?Phe?Asn?Thr?Thr?Tyr?Thr?Asp?Val?Arg?Ser?Thr?Asp?Trp?Phe?Ala
90 95 100
ggt?ttt?gta?aac?gta?gct?tcc?ggt?gaa?gaa?atc?gta?aaa?ggt?ttc?ccg 991
Glv?Phe?Val?Asn?Val?Ala?Ser?Gly?Glu?Glu?Ile?Val?Lys?Gly?Phe?Pro
105 110 115
gac?aaa?tct?ttt?aaa?cca?caa?aac?caa?gtt?act?tat?gct?gaa?gca?gta 1039
Asp?Lys?Ser?Phe?Lys?Pro?Gln?Asn?Gln?Val?Thr?Tyr?Ala?Glu?Ala?Val
120 125 130
acc?atg?atc?gtt?cgt?gct?ttg?ggt?tat?gag?cca?tcc?gtt?cga?ggt?gta 1087
Thr?Met?Ile?Val?Arg?Ala?Leu?Gly?Tyr?Glu?Pro?Ser?Val?Arg?Gly?Val
135 140 145
tgg?ccg?aac?agc?atg?atc?tcc?aaa?ggt?tcc?gaa?ctg?aac?att?gca?aaa 1135
Trp?Pro?Asn?Ser?Met?Ile?Ser?Lys?Gly?Ser?Glu?Leu?Asn?Ile?Ala?Lys
150 155 160 165
ggt?atc?aac?aac?cct?aac?atg?cag?cag?ttc?gcg?gcg?aca?atc?ttc?aaa 1183
Gly?Ile?Asn?Asn?Pro?Asn?Met?Gln?Gln?Phe?Ala?Ala?Thr?Ile?Phe?Lys
170 175 180
atg?ctg?gac?aac?gct?ctt?cgc?gtt?aag?ctg?atg?gag?caa?atc?gaa?tac 1231
Met?Leu?Asp?Asn?Ala?Leu?Arg?Val?Lys?Leu?Met?Glu?Gln?Ile?Glu?Tyr
185 190 195
ggt?act?gac?atc?cgt?tta?aac?gta?act?gac?gaa?act?ctc?ttg?act?aaa 1279
Gly?Thr?Asp?Ile?Arg?Leu?Asn?Val?Thr?Asp?Glu?Thr?Leu?Leu?Thr?Lys
200 205 210
tat?ttg?aaa?gtt?acc?gta?cgt?gat?atg?gac?tgg?gct?cac?gaa?aag?ggt 1327
Tyr?Leu?Lys?Val?Thr?Val?Arg?Asp?Met?Asp?Trp?Ala?His?Glu?Lys?Gly
215 220 225
aac?aat?tct?gat?gaa?ttg?cca?ctt?gta?aca?aac?gta?cct?gct?att?ggt 1375
Asn?Asn?Ser?Asp?Glu?Leu?Pro?Leu?Val?Thr?Asn?Val?Pro?Ala?Ile?Gly
230 235 240 245
ctg?ggt?agt?ttg?aaa?gca?aat?gaa?gtt?act?ttg?aat?gga?aaa?gat?gct 1423
Leu?Gly?Ser?Leu?Lys?Ala?Asn?Glu?Val?Thr?Leu?Asn?Gly?Lys?Asp?Ala
250 255 260
gat?ctg?ggt?agc?aac?act?act?tat?aaa?gta?gct?gaa?ggc?atc?aat?cct 1471
Asp?Leu?Gly?Ser?Asn?Thr?Thr?Tyr?Lys?Val?Ala?Glu?Gly?Ile?Asn?Pro
265 270 275
aac?gca?ttt?gat?ggt?caa?aaa?gta?caa?gtg?tgg?atc?aaa?gat?gac?cga 1519
Asn?Ala?Phe?Asp?Gly?Gln?Lys?Val?Gln?Val?Trp?Ile?Lys?Asp?Asp?Arg
280 285 290
gaa?aat?gtc?atc?gtt?tgg?atg?gaa?ggt?tcc?gaa?gac?gaa?gat?gtc?gtt 1567
Glu?Asn?Val?Ile?Val?Trp?Met?Glu?Gly?Ser?Glu?Asp?Glu?Asp?Val?Val
295 300 305
atg?gac?cgt?gtg?agt?gct?ctg?tac?ctg?aaa?ggt?aaa?gcc?ttc?aca?gat 1615
Met?Asp?Arg?Val?Ser?Ala?Leu?Tyr?Leu?Lys?Gly?Lys?Ala?Phe?Thr?Asp
310 315 320 325
gat?att?gta?aaa?gat?ctt?agc?aag?tct?gat?ttg?gat?gat?gta?aaa?atc 1663
Asp?Ile?Val?Lys?Asp?Leu?Ser?Lys?Ser?Asp?Leu?Asp?Asp?Val?Lys?Ile
330 335 340
gaa?atg?gat?ggt?agc?gag?aaa?tcg?tat?cgc?ttg?acg?gaa?gat?acg?aaa 1711
Glu?Met?Asp?Gly?Ser?Glu?Lys?Ser?Tyr?Arg?Leu?Thr?Glu?Asp?Thr?Lys
345 350 355
atc?act?tat?aac?ttc?act?cgt?ttc?aac?gat?cca?gta?gat?gct?ctg?agc 1759
Ile?Thr?Tyr?Asn?Phe?Thr?Arg?Phe?Asn?Asp?Pro?Val?Asp?Ala?Leu?Ser
360 365 370
aaa?att?tac?aaa?gac?aac?gac?acg?ttt?ggt?gtt?aaa?gtt?gtt?ttg?aat 1807
Lys?Ile?Tyr?Lys?Asp?Asn?Asp?Thr?Phe?Gly?Val?Lys?Val?Val?Leu?Asn
375 380 385
gat?aac?aat?gaa?gtt?gca?tac?ctc?cac?atc?att?gac?gat?caa?aca?att 1855
Asp?Asn?Asn?Glu?Val?Ala?Tyr?Leu?His?Ile?Ile?Asp?Asp?Gln?Thr?Ile
390 395 400 405
gat?aaa?agc?gta?aaa?ggc?gtt?aaa?tac?ggt?tcc?aaa?gtt?att?agc?aaa 1903
Asp?Lys?Ser?Val?Lys?Gly?Val?Lys?Tyr?Gly?Ser?Lys?Val?Ile?Ser?Lys
410 415 420
atc?gat?gct?gat?aag?aag?aaa?atc?acc?aac?ttg?gat?aac?tcc?aaa?ttt 1951
Ile?Asp?Ala?Asp?Lys?Lys?Lys?Ile?Thr?Asn?Leu?Asp?Asn?Ser?Lys?Phe
425 430 435
agc?gac?ttg?gaa?gat?cag?gat?gaa?gga?aaa?gac?ttc?ttg?gta?ttc?ctt 1999
Ser?Asp?Leu?Glu?Asp?Gln?Asp?Glu?Gly?Lys?Asp?Phe?Leu?Val?Phe?Leu
440 445 450
gat?ggc?caa?cca?gct?aaa?ctg?ggt?gat?ctg?aaa?gaa?tcc?gat?gtt?tac 2047
Asp?Gly?Gln?Pro?Ala?Lys?Leu?Gly?Asp?Leu?Lys?Glu?Ser?Asp?Val?Tyr
455 460 465
tcg?gta?tac?tat?gct?gat?ggt?gat?aaa?gat?aag?tac?ttg?gtg?ttt?gct 2095
Ser?Val?Tyr?Tyr?Ala?Asp?Gly?Asp?Lys?Asp?Lys?Tyr?Leu?Val?Phe?Ala
470 475 480 485
aac?cgt?aac?gtt?gca?gaa?gga?aaa?gta?gaa?aaa?gtg?gta?agc?cgt?aac 2143
Asn?Arg?Asn?Val?Ala?Glu?Gly?Lys?Val?Glu?Lys?Val?Val?Ser?Arg?Asn
490 495 500
aag?aca?gac?att?cgt?ctg?act?gtt?ggt?ggt?aaa?act?tac?aaa?gta?tat 2191
Lys?Thr?Asp?Ile?Arg?Leu?Thr?Val?Gly?Gly?Lys?Thr?Tyr?Lys?Val?Tyr
505 510 515
cca?gat?gct?tct?tac?tcc?gaa?aac?gca?aat?aaa?gat?gtt?aag?aag?gtt 2239
Pro?Asp?Ala?Ser?Tyr?Ser?Glu?Asn?Ala?Asn?Lys?Asp?Val?Lys?Lys?Val
520 525 530
aat?tcc?gac?ttg?gat?ctg?att?agc?aac?ctt?gat?gga?gaa?gaa?gta?aaa 2287
Asn?Ser?Asp?Leu?Asp?Leu?Ile?Ser?Asn?Leu?Asp?Gly?Glu?Glu?Val?Lys
535 540 545
ctt?ctc?ttg?gat?cca?tcc?ggt?cga?gtt?cgt?cac?atc?gaa?aca?aaa?gac 2335
Leu?Leu?Leu?Asp?Pro?Ser?Gly?Arg?Val?Arg?His?Ile?Glu?Thr?Lys?Asp
550 555 560 565
gcg?atc?gat?gat?cgt?aaa?cca?ttg?gca?atc?atc?act?aag?ggt?gct?acc 2383
Ala?Ile?Asp?Asp?Arg?Lys?Pro?Leu?Ala?Ile?Ile?Thr?Lys?Gly?Ala?Thr
570 575 580
tat?aac?tcc?agc?aaa?gat?act?tat?gac?ttt?act?gtt?atg?acc?caa?aaa 2431
Tyr?Asn?Ser?Ser?Lys?Asp?Thr?Tyr?Asp?Phe?Thr?Val?Met?Thr?Gln?Lys
585 590 595
ggt?aaa?aca?caa?atc?gta?tct?ttg?gat?caa?aag?gac?atc?tac?gat?aga 2479
Gly?Lys?Thr?Gln?Ile?Val?Ser?Leu?Asp?Gln?Lys?Asp?Ile?Tyr?Asp?Arg
600 605 610
tat?ggg?gtt?aac?tac?gat?aaa?tcc?aat?gat?aag?cgt?caa?gct?ttc?gaa 2527
Tyr?Gly?Val?Asn?Tyr?Asp?Lys?Ser?Asn?Asp?Lys?Arg?Gln?Ala?Phe?Glu
615 620 625
aag?gat?tta?gtt?gag?ctt?ctg?caa?cct?aag?gta?gtt?aaa?gag?gac?tca 2575
Lys?Asp?Leu?Val?Glu?Leu?Leu?Gln?Pro?Lys?Val?Val?Lys?Glu?Asp?Ser
630 635 640 645
gct?act?gat?gcc?aat?caa?acc?gtt?ctt?ttg?gaa?gtt?aat?ttt?gat?tcc 2623
Ala?Thr?Asp?Ala?Asn?Gln?Thr?Val?Leu?Leu?Glu?Val?Asn?Phe?Asp?Ser
650 655 660
aaa?ggc?gaa?gtt?gat?aaa?gtc?aaa?gtt?ctt?gat?agc?aag?ctg?aaa?tac 2671
Lys?Gly?Glu?Val?Asp?Lys?Val?Lys?Val?Leu?Asp?Ser?Lys?Leu?Lys?Tyr
665 670 675
tcc?gag?aaa?agc?act?tgg?gat?aaa?ctt?gcg?gat?gaa?gat?gac?gac?gtc 2719
Ser?Glu?Lys?Ser?Thr?Trp?Asp?Lys?Leu?Ala?Asp?Glu?Asp?Asp?Asp?Val
680 685 690
gtt?ggt?gat?tat?gaa?gta?acc?gat?aag?act?gct?gtc?ttc?aaa?atg?act 2767
Val?Gly?Asp?Tyr?Glu?Val?Thr?Asp?Lys?Thr?Ala?Val?Phe?Lys?Met?Thr
695 700 705
ggt?gac?ctt?act?cca?gct?act?gga?act?aaa?cgt?ggt?gaa?ctg?aaa?aac 2815
Gly?Asp?Leu?Thr?Pro?Ala?Thr?Gly?Thr?Lys?Arg?Gly?Glu?Leu?Lys?Asn
710 715 720 725
gct?ggt?act?gct?aaa?ttt?aag?gat?gtt?gct?aag?aag?agc?gat?ctg?aag 2863
Ala?Gly?Thr?Ala?Lys?Phe?Lys?Asp?Val?Ala?Lys?Lys?Ser?Asp?Leu?Lys
730 735 740
gtt?tgg?tac?tca?gtg?gat?gaa?gac?aaa?ggt?gaa?gtt?caa?gcg?atc?ttc 2911
Val?Trp?Tyr?Ser?Val?Asp?Glu?Asp?Lys?Gly?Glu?Val?Gln?Ala?Ile?Phe
745 750 755
gta?gta?gat?ggc?agt?ggt?ttg?ggt?ggc?gac?cat?cag?ttt?ggt?atg?gtc 2959
Val?Val?Asp?Gly?Ser?Gly?Leu?Gly?Gly?Asp?His?Gln?Phe?Gly?Met?Val
760 765 770
aaa?caa?tat?ggt?act?gct?agt?aaa?cag?gac?act?atc?act?ata?gtt?aca 3007
Lys?Gln?Tyr?Gly?Thr?Ala?Ser?Lys?Gln?Asp?Thr?Ile?Thr?Ile?Val?Thr
775 780 785
aaa?gat?ggt?gat?tct?gtc?act?gaa?aaa?gag?tac?aaa?ttg?gat?ggc?gac 3055
Lys?Asp?Gly?Asp?Ser?Val?Thr?Glu?Lys?Glu?Tyr?Lys?Leu?Asp?Gly?Asp
790 795 800 805
gct?gat?gat?ttg?aaa?gtt?gat?caa?gac?atc?aga?cgt?gga?gat?gtc?att 3103
Ala?Asp?Asp?Leu?Lys?Val?Asp?Gln?Asp?Ile?Arg?Arg?Gly?Asp?Val?Ile
810 815 820
tct?ttc?aca?ctg?aat?tct?gat?gga?gaa?gtt?att?gtt?gac?gat?gta?gtc 3151
Ser?Phe?Thr?Leu?Asn?Ser?Asp?Gly?Glu?Val?Ile?Val?Asp?Asp?Val?Val
825 830 835
gag?gtt?gta?aat?aac?aac?cac?att?gat?aac?aet?gct?tct?aaa?tca?gct 3199
Glu?Val?Val?Asn?Asn?Asn?His?Ile?Asp?Asn?Thr?Ala?Ser?Lys?Ser?Ala
840 845 850
acg?ctc?atg?cct?gaa?gac?gaa?cgt?caa?aaa?gca?gga?atc?gac?aaa?ttg 3247
Thr?Leu?Met?Pro?Glu?Asp?Glu?Arg?Gln?Lys?Ala?Gly?Ile?Asp?Lys?Leu
855 860 865
gtt?gtt?gct?cgc?gtt?gac?gaa?gtt?gat?ggt?aac?act?att?tcc?ttg?aac 3295
Val?Val?Ala?Arg?Val?Asp?Glu?Val?Asp?Gly?Asn?Thr?Ile?Ser?Leu?Asn
870 875 880 885
tat?gct?gac?gga?aag?aca?caa?aaa?tat?tac?aca?aaa?gca?tcc?act?gcg 3343
Tyr?Ala?Asp?Gly?Lys?Thr?Gln?Lys?Tyr?Tyr?Thr?Lys?Ala?Ser?Thr?Ala
890 895 900
ttc?att?gat?gtg?tat?gac?ggt?ctt?gaa?gga?att?gat?gga?gta?gat?gaa 3391
Phe?Ile?Asp?Val?Tyr?Asp?Gly?Leu?Glu?Gly?Ile?Asp?Gly?Val?Asp?Glu
905 910 915
ggc?gac?tac?atc?gta?atg?atc?gat?agc?gcc?gat?att?gac?gga?act?cgc 3439
Gly?Asp?Tyr?Ile?Val?Met?Ile?Asp?Ser?Ala?Asp?Ile?Asp?Gly?Thr?Arg
920 925 930
ttt?gac?tat?gta?ctg?gta?gtt?tct?agc?gat?gat?gag?atc?cgc?acg?caa 3487
Phe?Asp?Tyr?Val?Leu?Val?Val?Ser?Ser?Asp?Asp?Glu?Ile?Arg?Thr?Gln
935 940 945
cac?atc?tcc?act?aaa?gca?gtt?acg?gac?ttc?ctg?aac?aag?cca?acc?aga 3535
His?Ile?Ser?Thr?Lys?Ala?Val?Thr?Asp?Phe?Leu?Asn?Lys?Pro?Thr?Arg
950 955 960 965
cta?tgt?acc?aaa?tcc?tgg?cga?tgg?gga?aga?agt?agt?cac?ggc?acc?aaa 3583
Leu?Cys?Thr?Lys?Ser?Trp?Arg?Trp?Gly?Arg?Ser?Ser?His?Gly?Thr?Lys
970 975 980
gtt?aat?aca?gtt?aac?gat?gaa?gca?gtt?gta?gat?ggt?att?gta?act?ctt 3631
Val?Asn?Thr?Val?Asn?Asp?Glu?Ala?Val?Val?Asp?Gly?Ile?Val?Thr?Leu
985 990 995
cca?gct?gat?gca?tct?gtt?aga?aac?ttc?aac?att?gca?ttt?gat?caa?gaa 3679
Pro?Ala?Asp?Ala?Ser?Val?Arg?Asn?Phe?Asn?Ile?Ala?Phe?Asp?Gln?Glu
1000 1005 1010
att?aac?agc?aaa?gat?gca?acg?gta?act?gtt?act?aat?gaa?gat?acg?ctt 3727
Ile?Asn?Ser?Lys?Asp?Ala?Thr?Val?Thr?Val?Thr?Asn?Glu?Asp?Thr?Leu
1015 1020 1025
ggt?aac?gta?acg?gta?tct?gag?gtt?gcg?aca?gat?gca?aaa?gta?ttg?agc 3775
Gly?Asn?Val?Thr?Val?Ser?Glu?Val?Ala?Thr?Asp?Ala?Lys?Val?Leu?Ser
1030 1035 1040 1045
ttc?aag?act?gct?aaa?ctg?gac?act?aca?aaa?act?tac?atc?atc?aca?gtt 3823
Phe?Lys?Thr?Ala?Lys?Leu?Asp?Thr?Thr?Lys?Thr?Tyr?Ile?Ile?Thr?Val
1050 1055 1060
aaa?ggc?ttg?aaa?gat?aaa?aac?ggt?aaa?gca?gtg?aaa?gat?gta?act?ctt 3871
Lys?Gly?Leu?Lys?Asp?Lys?Asn?Gly?Lys?Ala?Val?Lys?Asp?Val?Thr?Leu
1065 1070 1075
tac?gtt?gaa?ttt?gtt?gct?gga?gtg?tgacttgttg?ctctataaac?actaatagtg 3925
Tyr?Val?Glu?Phe?Val?Ala?Gly?Val
1080 1085
tgaaatgagt?gaagaaggga?gaaggattaa?aaccttctcc?cttctttaac?tgttttggcg 3985
aattgaaggg?tgtaaccccc?ttccgtggaa?acaaccatta?atccacgtga?ctaatcctcc 4045
tgcatgcgtt?gtaagctaac?tcttacaggg?tgtgaagcca?ggtgaagacg?taacccttag 4105
tggtgcgggt?ctccgtaaaa?gctaactatg?gttaggaaac?gaatctacgt?ctgaggcgtg 4165
tcaggtttgg?taattagccc?ttcagctgct?aattacccca?caggctgaga?tgcctggagg 4225
gttctaacga?attaaggttg?ttaacgttag?ttcatcttga?tatgtatagt?aggaacctaa 4285
ggtttagcaa?aaaaagggat?agccaaaatg?gaacaagtga?gatct 4330
<210>42
<211>11
<212>DNA
＜213〉bacillus pumilus (Bacillus brevis)
<300>
<303>J.Bacteriol.
<304>169
<306>1239-1245
<307>1987
<400>42
agaggaggag?a 11
<210>43
<211>12
<212>DNA
＜213〉bacillus pumilus (Bacillus brevis)
<300>
<303>J.Bacteriol.
<304>169
<306>1239-1245
<307>1987
<400>43
gaaaggaggt?ga 12
Claims (18)
1. a kind of DNA of fusion rotein encodes, this fusion rotein is linked in sequence by following component and forms: begin the leading peptide (Leader) that is made of an above amino-acid residue from bacillus cell wall protein (CWP) N-terminal, be used for the aminoacid sequence (Cleavage) that enzyme is cut, and the aminoacid sequence (GH) of tethelin or its functional fragment, varient or analogue, and the aminoacid sequence of Cleavage is Ile-Glu-Gly-Arg (Cleavage 1, sequence numbering 1).
2. a kind of DNA of fusion rotein encodes, this fusion rotein is linked in sequence by following component and forms: begin the leading peptide (Leader) that is made of an above amino-acid residue from bacillus cell wall protein (CWP) N-terminal, be used for the aminoacid sequence (Cleavage) that enzyme is cut, and the aminoacid sequence (GH) of tethelin or its functional fragment, varient or analogue, and GH is 20kd human growth hormone (GH20kd).
3. a kind of DNA of fusion rotein encodes, this fusion rotein is linked in sequence by following component and forms: begin the leading peptide (Leader) that is made of an above amino-acid residue from bacillus cell wall protein (CWP) N-terminal, the nH that a plurality of Histidines (His) residue constitutes, be used for the aminoacid sequence (Cleavage) that enzyme is cut, and the aminoacid sequence (GH) of tethelin or its functional fragment, varient or analogue.
4. the DNA of claim 3 record, wherein His residue number is 6.
Claim 1,3 or 4 the record DNA, wherein GH is 22kd human growth hormone (GH22kd) or 20kd human growth hormone.
6. the DNA of each record of claim 2 to 4, wherein Cleavage is Ile-Glu-Gly-Arg (Cleavae 1, sequence numbering 1) or Glu-Asn-Leu-Tyr-Phe-Gln-Gly (Cleavae 2, sequence numbering 2).
7. the DNA of each record of claim 1 to 6, its upstream that is coded in described Cleavage has further connected the fused protein of the joint that is made of an above amino-acid residue.
8. the DNA of claim 7 record, wherein said joint is Asp-Tyr-Asp-Ile-Pro-Thr-Thr (sequence numbering 3).
9. the DNA of each record of claim 1 to 8, wherein said CWP is intermediate cell wall-held protein [middle wall protein (MWP)].
10. the DNA of claim 9 record, wherein said leading peptide are the aminoacid sequence of N-terminal to 1～20 from MWP.
11. the DNA of each record of claim 1 to 10, it is to be linked in sequence with this by the DNA that comprises the essential promotor of genetic expression (Promotert) sequence, at the DNA (CWPsp) of the SD sequence (SD) in this promotor 3 ' terminal downstream and bacillus cell wall protein (CWP) signal peptide of encoding in 5 ' the terminal upstream of the DNA of the above-mentioned fusion rotein of coding to form.
12. the DNA of claim 11 record, wherein above-mentioned promotor is the CWP from bacillus.
13. contain the carrier of the DNA of claim 11 or 12 records.
14. transform or the host cell of transfection with the carrier of claim 13 record.
15. the host cell of claim 14 record, it is a bacillus.
16. the host cell of claim 15 record, wherein above-mentioned bacillus is a bacillus pumilus.
17. the host cell of claim 16 record, wherein above-mentioned bacillus pumilus is for numbering the microorganism that FERM BP-7727 entrusts to declare.
18. the production method of recombinant human growth hormone or its functional fragment, varient or analogue, it is the production method of tethelin or its kinetic energy fragment, varient or analogue, comprise that the host cell of cultivating each record of claim 14 to 17 is expressed fusion rotein in substratum, cut out the operation of tethelin or its functional fragment, varient or analogue with enzyme process from this fusion rotein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001278534A JP2003079379A (en) | 2001-09-13 | 2001-09-13 | Dna for achieving high expression of growth hormone and use thereof |
| JP278534/2001 | 2001-09-13 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1547610A true CN1547610A (en) | 2004-11-17 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA028166523A Pending CN1547610A (en) | 2001-09-13 | 2002-09-09 | DNA for achieving high expression of growth hormone and use thereof |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP2003079379A (en) |
| CN (1) | CN1547610A (en) |
| WO (1) | WO2003025182A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102389575A (en) * | 2011-11-18 | 2012-03-28 | 武汉凯肽来生物科技有限公司 | Genetic engineering oral DNA vaccine and preparation method and application |
| CN107142235A (en) * | 2017-06-20 | 2017-09-08 | 江西嘉博生物工程有限公司 | A kind of restructuring Brevibacillus brevis for expressing pig growth hormone gene and construction method and application |
| CN109957028A (en) * | 2017-12-22 | 2019-07-02 | 丰益(上海)生物技术研发中心有限公司 | Improve the method that the mature peptide of secreted protein generates |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2251425B1 (en) | 2004-07-06 | 2016-04-20 | Kaneka Corporation | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1251895B (en) * | 1991-09-27 | 1995-05-26 | Eniricerche Spa | HUMAN GROWTH HORMONE MUTANTS AND THEIR USE |
| JP3313083B2 (en) * | 1998-03-31 | 2002-08-12 | 伊藤ハム株式会社 | DNA coding for novel fusion protein and method for producing useful polypeptide via its expression |
-
2001
- 2001-09-13 JP JP2001278534A patent/JP2003079379A/en active Pending
-
2002
- 2002-09-09 WO PCT/JP2002/009155 patent/WO2003025182A1/en active Application Filing
- 2002-09-09 CN CNA028166523A patent/CN1547610A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102389575A (en) * | 2011-11-18 | 2012-03-28 | 武汉凯肽来生物科技有限公司 | Genetic engineering oral DNA vaccine and preparation method and application |
| CN107142235A (en) * | 2017-06-20 | 2017-09-08 | 江西嘉博生物工程有限公司 | A kind of restructuring Brevibacillus brevis for expressing pig growth hormone gene and construction method and application |
| CN109957028A (en) * | 2017-12-22 | 2019-07-02 | 丰益(上海)生物技术研发中心有限公司 | Improve the method that the mature peptide of secreted protein generates |
| CN109957028B (en) * | 2017-12-22 | 2023-04-07 | 丰益(上海)生物技术研发中心有限公司 | Methods for increasing production of mature peptides of secreted proteins |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2003079379A (en) | 2003-03-18 |
| WO2003025182A1 (en) | 2003-03-27 |
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