CN101613687A - Recombination ox intestine kinase, Preparation Method And The Use - Google Patents

Abstract

The invention discloses recombination ox intestine kinase light chain mutant and recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein, wherein the heavy chain structural domain is connected by small peptide with the light chain mutant, the present invention further discloses aminoacid sequence, its preparation method and its purposes as the fusion rotein cutting agent of said mutation body and fusion rotein.Above-mentioned recombination ox intestine kinase light chain mutant disclosed by the invention and recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein can obtain by simple purification procedures, have active high advantage.

Description

Recombination ox intestine kinase, Preparation Method And The Use

The application is to be on May 6th, 2008 applying date, and application number is 200810092899.9, and denomination of invention is divided an application for China of " recombination ox intestine kinase, Preparation Method And The Use " application.

Technical field

The invention belongs to biological technical field, more specifically, the invention discloses recombination ox intestine kinase mutant and recombination ox intestine kinase fusion rotein, its preparation method and application.

Background technology

(EP is to be present in the ID a kind of heterodimer of Mammals (heterodimeric) serine protease EC3.4.21.9) to enteropeptidase for Enterokinase, EK or Enteropeptidase.Molecular mass is 150kDa, forms by the heavy chain of 1 115kDa and the light chain of 1 35kDa, and at pH value 4.5-9.5, temperature 4-45 ℃ of scope internal specific protolysate substrate.Because the desired polypeptides that the EK crack fusion protein is discharged has and the on all four-terminal amino acid sequence of wild-type, thereby can be used as the cutting reagent of fusion rotein in the protein expression system.But natural enteropeptidase source is limited after all, and the cost height of extraction separation, and the enteropeptidase of natural extract easily is other proteolytic enzyme pollution, the purpose product of having degraded again when causing cleavage of fusion proteins in addition.And engineered method just can remedy these deficiencies, thereby uses engineered method to produce enteropeptidase and widely use.Commercial enteropeptidase mainly contains two kinds: the Enteropeptidase of natural purification and gene engineering recombinant cattle enteropeptidase.

Enteropeptidase has following characteristics:

Constructional feature: the natural intestine kinases is made of 1 structure subunit (heavy chain) and 1 catalytic subunit (light chain), both are by 1 intermolecular disulfide bond combination, the structure subunit is responsible for being fixed on catalytic subunit on the intestinal brush border film and guiding it to move to enteric cavity, catalytic subunit can specific recognition Asp-Asp-Asp-Asp-Lys sequence and is downcut along the carboxyl terminal of sequence, the trypsinogen activation is trypsinase, thereby starts the cascade of various proenzyme activatory.

Recombinant enterokinase (recombinant Enterokinase, molecular mass rEK) is generally 26.3kDa, has 3 glycosylation sites, and its glycosylation molecular mass is approximately 43kDa.Discoveries such as Vozza, rEK experiment in vitro proof have the holoenzyme enzyme to cut specificity and show compared to the natural intestine kinases enzyme of genetic engineering fusion protein substrate is cut increased activity, thereby now adopt engineered method to produce enteropeptidase more.Mostly the trial of being done in recent years is 235 amino acid whose light chains of cloning and expression (recombinantEnterokinase light chain, rEK _L).It is amino that Janska etc. study, and terminal residue has been found the structural homology of the serine protease of the three-dimensional structure of EK light chain and tryptase etc.Seong etc. find that then the aminoterminal Ile of light chain is sported its enzymic activity of Val to change hardly.In addition, there is the heavy chain of discovering EK to influence enteropeptidase strongly to the identification of macromolecule substrate and to the not influence of identification of synthetic fusion rotein or small molecules substrate.

The zymetology characteristics: enteropeptidase activates trypsinogen in vivo and is converted into trypsinase, because the light chain structure of enteropeptidase is the people, conservative in ox and the pig, its recognition sequence Asp-Asp-Asp-Asp-Lys also has very strong conservative property in vertebrates, and nearly all had the feature that 4 l-asparagines link to each other by the trypsinogen of sequencing, this sequence is very rare again on other natural protein, and there is 1 special positively charged ion site in the active centre of enteropeptidase, make the Asp-Asp-Asp-Asp positively charged ion site combination therewith of powerful negative electricity is arranged, therefore, the substrates enzymes of Enteropeptidase is cut the specificity that site sequence has height, and this characteristics make EK become genetic engineering fusion protein and express in the modification of back 1 exceedingly useful instrument and be widely used.For the high degree of specificity that the EK enzyme is cut, Rumsh thinks to make enzyme from cutting owing to the forfeiture of calcium ion in the enzyme, thereby has possessed the high degree of specificity that activates other enzyme.But in the endonuclease reaction of reality, also may produce some nonspecific cuttings, in genetically engineered research, should take seriously.

Separation and purification characteristics: similar with most proteic separation purification method, rEK can obtain pure product by methods such as ammonium sulfate precipitation, DEAE post, gel chromatography and dialysis, in addition, utilization Histidine (Histidine, His) carry out mark at the albumen end, the method of protein isolate of this one step of low-cost high-efficiency of Nickel metal chelating column affinity purification also applies to the separation and purification of rEK just widely, and its yield can reach more than 50%.For the site of His-tag, Choi etc. discover at rEK _LC-terminal carry out the His mark, do not change with its enzymic activity behind the Nickel metal chelating column affinity purification, after N end carries out the His mark by the then enzymic activity forfeiture of Nickel metal chelating column.

Physilogical characteristics: nineteen thirty-nine Kunitz confirms that enteropeptidase is the physiology activator of trypsinogen, and trypsinase is the activator of other many proenzymes of Digestive tract, so enteropeptidase is considered to one of important initial enzyme of Digestive tract.It is generally acknowledged that EK is present on the intestinal brush border cytolemma, Zamolodchikova etc. think that pro-EK is by the preduodenal lipases activated.Pathological study is found, thereby enrichment EK can activate too much trypsinogen and causes acute pancreatitis in the phegma of duodenum and pancreas, and the body of EK defective will have symptoms such as diarrhoea, vomiting, edema, cause dysplasia, cause not enough disease of haemproteins and anaemia.

The genetically engineered progress of EK

Expression in prokaryotic organism: the genetically engineered in the procaryotic cell expression system is to adopt intestinal bacteria Escherichiacoli to be used as the host bacterium mostly.But should consider the exactness and the proteic activity of expressed proteins disulfide linkage for the albumen that expression contains disulfide linkage, thereby for guaranteeing the correct formation of enteropeptidase disulfide linkage, scholars adopt methods such as merging DsbA, thioredoxin to make the enteropeptidase of expressing that activity be arranged.LaVallie etc. have adopted amalgamation and expression companion DsbA protein secreting, expressing EK in Escherichia coli _LWith ox EK _LThe cDNA sequence merges 3 ' end in the DsbA sequence, and the sequence of passing through the specific recognition site of coding EK between fusion rotein links to each other, and just can obtain activated rEK by cutting processing certainly _L, and the ability that its cutting contains the fusion rotein of Asp-Asp-Asp-Asp-Lys sequence is better than the natural intestine kinases.Huang He etc. are by extracting the ox total tissue RNA, obtain ox EKL cDNA with wealthy the increasing of RT-PCR method, this gene being inserted make up ox EK among the pET39b _LFusion expression vector pET39b-EK _L, in Escherichia coli BL21 (DE3), induce and express with IPTG, separate by one step of Nickel metal chelating column, obtained activated enteropeptidase.Yuan etc. are with ox EK _LThe cDNA sequence merges in amalgamation and expression companion thioredoxin (Trx) downstream, expresses at Escherichia coli BL21 (DE3), in vivo autocatalysis cleavage of fusion proteins Trx-EK _LAfter, complete bioactive rEK is arranged _LBe released, through method purifying such as DEAE, survey it and be 720Aus/mg than living, Km=0.17mM, K (cat)=20.8s/L, 100ml shake bottle and obtain 4.3mg EK _LPure product.Similarly, Gasparian etc. is with people EK _LThe cDNA sequence merges in the Trx downstream, be cloned on the pET32a, express at Escherichia coli BL2J (DE3), the product of solubility does not have from cutting active, from inclusion body, obtained activated recombinant human enteropeptidase catalyzing subunit (L-HEP) by solubilising and renaturation, with the L-HEP cutting Gly-Asp-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide of purifying, its Km=0.16Mm, K (cat)=115s/L.Under the same conditions, the speed of L-HEP digestion Trx-human epidermal growth factor Trx-hEGF (thioredoxinhumanepidermal growth fator) is than the ox EK of identical activity unit _LFast 5 times.

Expression in eukaryote is included in fungi, mammalian cell and the yeast and expresses, and will insert KEX-2 proteolytic enzyme enzyme and be cut to a little ox EK for expressing Svetina etc. in the fungi therein _LCDNA merge in glucoamylase (glucoamylase) gene downstream, in aspergillus niger (filamentous fungus Aspergillus niger AB1.13), carry out secreting, expressing, fusion rotein is being processed into sophisticated EKL by endonuclease KEX-2 in cell Periplasmic secretion way, utilization ion exchange chromatography and affinity chromatography separation and purification nutrient solution supernatant can obtain activated EKL1.9mg/L, production peak at the soybean milk medium can reach 5mg/L (being higher than the output in E.coli), though be lower than the expression amount in methanol yeast, the yield 32ug/g of EKL is but much larger than the 12ug/g in methanol yeast in wet thallus.For animal cell expression, 1993, LaVallie ER etc. were fused to EK with the propetide of Mammals serine protease PACE as the secretion homing sequence _LN-terminal, with this fusion gene PACE-EK _LBe building up on the pMT3 expression vector, transfection monkey kidney COS-1 cell carries out secreting, expressing then, has obtained SA EK _L, discover by PACE-EK simultaneously _LThe EKL that/KEX2 coexpression obtains, the activity of its cutting fluorogenic substrate Gly-Asp-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide has enhanced trend.For utilizing yeast cell to express, the yeast that is usually used in the functional expression of foreign protein is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and methanol yeast (Pichia pastoris), these two systems all have the function of posttranslational modification: the formation of disulfide linkage, proteic glycosylation etc., these translation post-treatment mode comparing class are similar to mammalian cell.Because yeast can not utilize the secreting, expressing of the signal sequence guiding foreign protein of foreign gene, thereby Saccharomyces cerevisiae and Pichia pastoris utilize the secretion signal-mating factor of the yeast saccharomyces cerevisiae of being made up of 89 amino acid, and (MF) leader sequence guides the secreting, expressing of foreign protein.Compare with other protein expression system, the advantage of yeast expression system is: the strict expression of transferring empty foreign protein of strong AOX1 promotor is safe to the people, can in Routine Test Lab, use and expense cheap.

Ox EK _L4 pairs of intramolecular disulfide bonds are arranged, and can S-S correctly form the direct EK of influence _LWhether activity is arranged, thereby the characteristics that Chinese scholars utilizes yeast can translate post-treatment are one after another expressed EK at Yeast system _LAlbumen.

The trial of being done at Saccharomyces cerevisiae system expression enteropeptidase is that 235 the amino acid whose ox EKL cDNA that will encode such as Choi and Seong are cloned into pIL20Gh, makes up pIL20EK _LSecretion expression carrier ,-MF are promotor, with the people-24 amino acid of interleukin (interleukin-1) N-terminal are as the secretion enhanser, and at interleukin-1 and EK _LBetween introduced the KEX restriction enzyme site, carry out secreting, expressing at Saccharomyces cerevisiae 2805, product obtains activated EK through the separation and purification of Nickel metal-chelating _L(about 1mg/L).

Pichia pastoris expression system since being applied to single cell protein and producing the seventies, through nearly 20 years development, the expression system of kind of the foreign protein that developed into a successful expression surplus in the of 200.PA0804, pA0815 and pPIC3 etc. are its expression vector commonly used.The maximum characteristics of Pichia system: the secretion level of native protein is extremely low, and making becomes main component in the substratum behind the exogenous protein expression.Therefore, utilize Pichia pastoris system expression foreign protein to be widely used in the genetically engineered field.Grandson waits the EK that increases with RT-PCR from brave _LCdna, make up pPICZaA-EK _LExpression vector, secreting, expressing in host Picha pastoris GS115, fermented liquid can obtain EK behind the Zn-Sepharose affinity purification _L1.6mg/L (purity is greater than 90%), yield are higher than the purifying 1mg EK from 1L cereuisiae fermentum nutrient solution of Choi report _LYield, it is to Km=753 ± 42mM of small molecules fluorogenic substrate Gly-Asp-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, K (cat)=31.5 ± 3.8s/L.LFang etc. are incorporated into ox EKL in the pA0815 plasmid, always secrete expression with methanol induction at host Pichia pastoris GS115, obtain activated EK through Nickel metal-chelating column separating purification fermentation supernatant _L5.4mg/L.Vozza etc. with-MF is leader sequence secreting, expressing ox EK in methanol yeast _L, the purified fermentation broth supernatant obtains activated EK _L6.3mg/L expressing quantity is apparently higher than the expression amount in E.coli and COS cells.2004, Peng Zhong methanol yeast expression system secreting, expressing EK _L, use glycerine/methyl alcohol substratum at pH value 6.0, rEK _LOutput reach 350mg/L, behind anion-exchange chromatography, obtain EK _LBe 15mg/L.

Although recombinant enterokinase has been carried out a large amount of research, the Enterpeptidase light chain inclusion body products that obtains for the escherichia expression system of present widespread use exists inclusion body products renaturation difficulty, the problem that protein-active is low, and, along with development of biology, still need the expression amount height, the active recombinant enterokinase of getting well and being applicable to production.

Summary of the invention

In order to address the above problem, the present inventor has carried out a large amount of experiments, with reference to Enteropeptidase light chain EKL and its inhibitor VD ₄The crystalline structure of the mixture of K-chloromethane (Code no:1EKB, Protein Dtabase Bank) is observed with VD ₄K is center 5 The situation of the amino-acid residue in the scope is selected may to strengthen EKL after the sudden change and is combined active residue with DDDDK, and it is sported R, to strengthen itself and VD ₄The bonding force of D among the K.Designed 9 mutant altogether, respectively called after EKLm1, EKLm2, EKLm3, EKLm4, EKLm5, EKLm6, EKLm7, EKLm8, EKLm9, determination of activity comparison shows that Michaelis-Menton constant Km and maximum reaction velocity Vmax and the R﹠amp of wild-type EKL; The D like product is similar, and in 9 mutant, the Km of EKLm3, EKLm5, EKLm6, EKLm7 and EKLm8 compares the decline that all has in various degree with wild-type, wherein reduces the most significantly with the Km value of EKLm6, and promptly EKLm6 has the avidity with substrate the best.

The present inventor also adopts pichia yeast expression system that EKLm6 is expressed, obtained highly active EKLm6, experimental result shows that the activity of the EKLm6 of yeast expression is higher than EKL too, and the EKLm6 activity of yeast expression is 2 times of same protein of escherichia coli expression.

Because the Enterpeptidase light chain inclusion body products renaturation difficulty that escherichia expression system obtains, so the yield of activated protein is extremely low.In order to improve the output of EKL activated protein as far as possible, each structural domain of choosing enteropeptidase heavy chain EKH respectively links to each other through the flexible Linker of one 10 peptide with EKL and constitutes fusion rotein, by abduction delivering EKL, EKLm6, EKH1-EKLm6, EKH3-EKLm6, EKH4-EKLm6, EKH5-EKLm6, the result shows that every 100g EKH5-EKLm6 inclusion body recoverability purifying obtains 113.8mg albumen, its yield is than EKL (10.4mg), EKLm6 (9.8mg), EKH1-EKLm6 (7.6mg), EKH3-EKLm6 (12.6mg) and EKH4-EKLm6 (9.5mg) are significantly increased, more specifically, in order to strengthen the renaturation effect, improve the yield of activated protein, PEG-8000 and glycerol in renaturation buffer, have been added, accessory protein correct folding and keep proteic activity.In addition, adopt the high STI affinity column of specificity to come the activated albumen of purifying, can effectively correctly folding target protein be separated with correct folding target protein and foreign protein, obtain purity and reach activated protein more than 98%, improve proteic purity greatly, avoided the more drawback of metal chelating column purifying protein impurity.

Determination of activity shows the active similar of the EKH5-EKLm6 fusion rotein that utilizes escherichia coli expression and EKLm6, with the existing R﹠amp in market; The product of D company is compared, and its activity has improved nearly 10 times, the active and existing R﹠amp in market of the EKLm6 of yeast expression; The product phase specific activity of D company has improved nearly 20 times.

The above results shows that recombination ox intestine kinase disclosed by the invention has reached purpose of the present invention.

Description of drawings

Fig. 1: the tomograph of the mixture of Enteropeptidase light chain and its inhibitor VD4K-chloromethane, wherein the Enteropeptidase light chain amino acid is represented with band; The amino-acid residue of VD4K-chloromethane identifies with light color; The amino-acid residue dark mark that closely contacts with inhibitor in the Enteropeptidase light chain;

Fig. 2: recombination ox intestine kinase light chain mutant sequence comparison diagram, deshed line represent in the mutant with EKL at the identical amino-acid residue of opposite position;

Fig. 3: recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein gene structure figure; 3-1:EKH1-EKLm6; 3-2:EKH3-EKLm6; 3-3:EKH4-EKLm6; 3-4:EKH5-EKLm6;

Fig. 4: EKL and mutant Km, Vmax measure, the Linewaver-Burk result that maps; Fig. 4-1:EKLm1; Fig. 4-2:EKLm2;

Fig. 4-3:C.EKLm3; Fig. 4-4:EKLm4; Fig. 4-5:EKLm5; Fig. 4-6:EKLm6; Fig. 4-7:EKLm7; Fig. 4-8:EKLm8; Fig. 4-9:EKLm9;

Fig. 5: temperature is cut the result that influences of thioredoxin fusion rotein Trx/hIL-11 to enzyme; Wherein 1:20 ℃, 2:10 ℃, 3:30 ℃;

Fig. 6: the reaction times is cut the result that influences of thioredoxin fusion rotein Trx/hTL-11 to enzyme; 1:40hr wherein, 2:20hr, 3:10hr, 4:5hr;

Fig. 7: the enzyme enzyme of different amounts is cut the result of thioredoxin fusion rotein Trx/hIL-11; 1:1.28ng wherein, 2:0.64ng, 3:0.32ng, 4:0.16ng, 5:0.08ng, 6:0.04ng, 7:0.02ng, 8:0;

The Detection of Stability result of Fig. 8: EKH5-EKLm6; Wherein 1:-20 ℃, to preserve 6 months, 2:-20 ℃ and 37 ℃ 3 times were repeatedly placed 1 day for 3:37 ℃, placed 3 days for 4:37 ℃, placed 5 days for 5:37 ℃, placed 7 days for 6:37 ℃, placed 8 10 days for 7:37 ℃: contrast;

Fig. 9: concentration of substrate is to the influence curve of enzymatic reaction speed;

Figure 10: two figure results that sit reciprocal.

Figure 11: temperature of reaction is cut the experimental result that influences of thioredoxin fusion rotein Trx-MBL-CLR to enzyme

Figure 12: the reaction times is cut the experimental result that influences of thioredoxin fusion rotein Trx-MBL-CLR to enzyme

Figure 13: enzyme is cut the optimal proportions experimental result of thioredoxin fusion rotein Trx-MBL-CLR

The Detection of Stability result of Figure 14: EKH5-EKLm6

Embodiment

Following examples only further specify the present invention, should not be construed as limitation of the present invention, embodiment does not comprise the detailed description to traditional method, be used for the method for carrier construction and plasmid as those, the gene of proteins encoded be inserted into the method for such carrier and plasmid or plasmid is introduced the method for host cell and classical cytogamy and screening, separated and method such as purifying.Such method is well-known for the person having ordinary skill in the art, and all describes to some extent in many publications, comprises Sambrook, J., Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning:A LaboratoryManual, 2 ^NdEdition, Cold spring Harbor Laboratory Press.

Former, the auxiliary material of not indicating the source in the embodiment of the invention are commercially available.Thioredoxin-human interleukin-11 (Trx/hIL-11) (Shanghai CP Guojian Pharmaceutical Co.,Ltd) Trx-MBL-CLR: prepare by the middle disclosed method of " structure of people MBL-CLR expression vector and the expression in intestinal bacteria thereof " (" cell and molecular immunology magazine " 2007 No.23 (1) Page:25-7,31 Cai Xue are quick, Zuo Daming etc.)

1 N of segmental clone of EK cDNA of embodiment

Get the fresh ox duodenum of 150mg, extract total RNA, get the total RNA of 0.5 μ l, add 3 μ l oligo (dT), 18 primer primers, mend and add water to cumulative volume 10.5, mixing with Trizol reagent (Invitrogen company product).Room temperature is placed 10min, high speed centrifugation 5min.Add 4.0 μ l, 5 * reverse transcription reaction damping fluid more successively, 0.5 μ l RNA enzyme inhibitors, 2.0 μ l 10mMdNTP, 2.0 μ l DTT, 1 μ l MMLV reversed transcriptive enzyme, mixing is put 37 ℃ of reaction 120min.Get 1.5 μ lRT products, adding 100pmol upstream primer (5 '-aagcttatggggtcaaagcgaagtgt-3 ') and downstream primer (5 '-gaattctcaatgtagaaaactttgtatcc-3 ', encoding sequence with reference to ox Enterokinase among the CenBank, sequence number: U09859 designs primer), 2.5 μ l, 10 * PCR damping fluid, 2 μ l 2.5mMdNTP, 2.5 DMSO, 0.25 the Taq enzyme adds water to final volume 25 μ l, the paraffin oil that adds 5 μ l behind the mixing rapidly carries out pcr amplification.Reaction conditions is 94 ℃ of sex change 4min; Carry out following 30 circulations then: 94 ℃ of sex change 50s, 51 ℃ of annealing 50s.72 ℃ of 3min; Extend 8mim at 72 ℃ at last.With cDNA fragment electrophoresis, carry out the glue Separation and Recovery according to the test kit specification sheets then.The PCR product that 1 μ l pMD18-T carrier and 8 μ l are reclaimed under the effect of T-4DNA ligase enzyme 16 ℃ be connected and spend the night, transform the competence escherichia coli DH5a of Calcium Chloride Method preparation then.Picking single white bacterium colony is 5 at random, and 37 ℃ are shaken bacterium and spend the night, and carries out plasmid and extracts, and cuts evaluation with HindIII and EcoRI enzyme, and the fragment of inserting is carried out complete sequence determination, filters out correct insertion person, called after pMD18-T-EK.

The structure of the design of embodiment 2 EKLm and pET39b-EKL and pET39b-EKLm expression plasmid

In order to improve the activity of enteropeptidase, the present inventor is with reference to Enteropeptidase light chain and its inhibitor VD ₄The crystalline structure of the mixture of K-chloromethane (Code no:1EKB, Protein Dtabase Bank) is observed with VD ₄K is center 5

The situation of the amino-acid residue in the scope is selected may to strengthen EKL after the sudden change and is combined active residue with DDDDK, and it is sported R, to strengthen itself and VD ₄The bonding force of D among the K.9 mutant have been designed altogether, as shown in Figure 1.

To identify that correct pMD18-T-EK is a template, the PCR Enteropeptidase light chain coding region cDNA fragment that increases once more, upstream primer is: 5-gaattcggacgacgacgacaagattgtcggaggaagtgactcc-3 ', downstream primer is: 5 '-aagctttcaatgtagaaaactttgtatcc-3 '.Upstream primer band EcoRI restriction enzyme site and enteropeptidase recognition site, downstream primer band HindIII restriction enzyme site and terminator codon.The pcr amplification condition is 94 ℃ of sex change 4min; Carry out following 30 circulations then: 94 ℃ of sex change 45s, 52 ℃ of annealing 45s.72 ℃ are extended 1min; Extend 7mim at 72 ℃ at last.The pcr amplification product agarose gel electrophoresis reclaims (method is the same) after identifying, makes up the EKL gene.On this basis, for improving it in conjunction with activity, do 9 mutant, the mutational site as shown in Figure 1, name respectively and be EKLm1, EKLm2, EKLm3, EKLm4, EKLm5, EKLm6, EKLm7, EKLm8, EKLm9, the mutant gene that order-checking is correct respectively with the pET39b plasmid with HindIII and 37 ℃ of double digestions of EcoRI 2 hours.Cut product through separation of 0.8% agarose gel electrophoresis and recovery enzyme.Purpose fragment and carrier mixed in 4: 1 in molar ratio, 10 μ l linked systems, and 1 μ l T4 dna ligase, 16 ℃ of water-baths are spent the night.Connect product Transformed E .coli BL21 (DE3) competent cell, select recombinant clone and cultivate, extract plasmid DNA, identify correct clone through HindIII and EcoRI double digestion, difference called after pET39b-EKL and pET39b-EKLm1, pET39b-EKLm2, pET39b-EKLm3, pET39b-EKLm4, pET39b-EKLm5, pET39b-EKLm6, pET39b-EKLm7, pET39b-EKLm8, pET39b-EKLm9.

The abduction delivering of embodiment 3 pET39b-EKL and pET39b-EKLm

The trioredoxin analog thing DsbA that provides on the pET39b carrier can help protein normally folding, thereby produces activated soluble protein.Each engineering bacteria is inserted in the LB substratum.Put into shaking table at 37 ℃, incubated overnight under the 250rpm condition.Seed liquor after will spending the night adds in the 2YT substratum, puts into shaking table at 37 ℃, cultivates under the 250rpm condition.Work as OD ₆₀₀Be 0.6 o'clock, add 0.1mM IPTG and induce, put into shaking table, continue under the 250rpm condition to cultivate at 30 ℃.Take a sample after inducing 2h, the centrifugal supernatant that goes, precipitation is put into refrigerator, and every then 1h of mistake takes a sample, and the same centrifugal supernatant that goes is put into refrigerator with precipitation.Receive bottle, the centrifugal supernatant that goes after inducing 6h.PAGE electrophoresis detection abduction delivering situation, the target protein expression amount is the highest when inducing 4 hours.The experimental result of expressing according to the intestinal bacteria shaking table inserts the activatory engineering bacteria in the LB substratum, puts into shaking table, at 37 ℃, and incubated overnight under the 250rpm condition.Seed liquor after will spending the night adds in the fermentor tank, 37 ℃ of culture temperature, and it is 7.4 that ammoniacal liquor is regulated pH, saturated oxygen gas integration number keeps＞130%.When OD was 8.0, contrast was done in sampling, and added 0.3mM IPTG and induce, and temperature is reduced to 30 ℃.Receive jar, the centrifugal supernatant that goes after inducing 3h.

The renaturation and the purifying of embodiment 4 EKL and mutant thereof

The escherichia coli fermented broth centrifugal (8000rpm, 10min, 4 ℃) of expressing EKL and mutant thereof is abandoned supernatant, with damping fluid (50mMTris-HCl pH8.5) with the 1.0g thalline: 10ml damping fluid suspension thalline.Treat to dissolve fully.Ultrasonic then (800w/30s, 4-6 time) breaks bacterium 30min, and after microscopy showed that thalline breaks fully, centrifugal (12000rpm, 15min, 4 ℃) abandoned supernatant, collected the inclusion body precipitation, were used to be further purified.Every gram inclusion body is with 8ml lysate (8M urea, 20mMTris 100mM 2-MT PH8.0) sex change dissolving, and dilution refolding (to urea final concentration 1M) spent the night for 16 ℃ after β-MT was removed in desalination.Desalination is to enzyme cutting buffering liquid (20mM Tris50mM NaCl 1mM CaCl ₂PH 8.75) autotomyed 10 hours.With STI (specificity pancreatin inhibitor, but specific combination EK) affinity chromatography column purification EKL and mutant thereof.Every 100g inclusion body recoverability purifying obtains about 5-15mg albumen.

The Michaelis-Menton constant (Km) of embodiment 5 EKL and mutant thereof and the mensuration of maximum reaction velocity (Vm)

Under temperature, pH and enzyme concn constant condition, concentration of substrate has very big influence to the speed of enzymatic reaction.When concentration of substrate was very low, speed of response is hurried quickening with the increase of concentration of substrate, both proportional relations; When concentration of substrate was higher, though speed of response is accelerated along with the rising of concentration of substrate, no longer proportional example was accelerated; When concentration of substrate was increased to a certain degree, if continue to strengthen concentration of substrate, speed of response no longer increased, and illustrated that enzyme is saturated by substrate institute.

Under the constant situation of the concentration of enzyme, concentration of substrate presents the rectangle hyperbolic line to the effect of speed of response influence.See Fig. 9.

Variation relation between enzymatic reaction speed and the concentration of substrate has reflected the formation and the process that generates product [P] of [ES].When [S] was very low, the active centre of enzyme did not all combine with substrate, increased [S], and the formation of [ES] increases with all proportional relation of the generation of [P]; When [S] increased to finite concentration, enzyme had all formed [ES], and increase [S] again and also can not increase [ES] this moment, and it is constant that speed of response is tending towards.

In order to explain the relation of concentration of substrate and enzymatic reaction speed, Michaelis in 1913 and Menten reduce the most basic mathematic(al) representation of enzyme kinetics---Michaelis-Menton equation Michaelis-Menten to last figure:

$v=\frac{V_{\max }\left[S\right]}{K_m+\left[S\right]}$

V in the formula---speed of response;

Km---Michaelis-Menton constant;

Vmax---enzyme reaction top speed;

[S]---concentration of substrate.

In enzymatic analysis, Km is an essential characteristic constant of enzyme, and it is comprising enzyme-to-substrate combination and dissociated character.Km and concentration of substrate, enzyme concn have nothing to do, and be relevant with factors such as pH, temperature, ionic strengths.For each enzymatic reaction, its specific Km value is all arranged under certain condition, therefore can be used for differentiating enzyme.

The meaning of Michaelis-Menton constant (Km):

When speed of response be top speed one half, Michaelis-Menton equation is can conversion as follows:

1/2Vmax＝Vmax[S]/(Km+[S])

So Km=[S].

Therefore, the Km value equals the concentration of substrate of enzymatic reaction top speed one half.

(the Km value is bigger, and the avidity of enzyme-to-substrate is littler 2.Km value can be judged the avidity of enzyme-to-substrate; Vice versa).

3.Km value is the characteristic constant of enzyme, and is only relevant with enzymatic reaction condition with structure, the catalytic substrate of enzyme institute of enzyme, irrelevant with the concentration of enzyme.The kind difference of enzyme, the Km value is different, and the same enzyme is during with different substrate-function, and the Km value is difference also.

Measure Km, Vmax, generally try to achieve with graphing method.Graphing method has a lot, and the most frequently used is the Linewaver-Burk graphing method, and this method is the form reciprocal according to Michaelis-Menton equation, with 1/v to 1/[S] mapping, can obtain straight line (seeing figure).The intercept of straight line on transverse axis is-1/Km that vertical intercept is 1/Vmax, can obtain Km and Vmax.

$\frac{1}{v}=\frac{K_m}{V_{\max }}\·\frac{1}{\left[S\right]}+\frac{1}{V_{\max }}$

See Figure 10.

Enterokinase is active to be measured by its fluorogenic substrate Gly-Asp-Asp-Asp-Asp-Lys-naphthylamide (Sigma).

Peptide substrate is dissolved in and contains 10%DMSO, and 70mM Tris-HCl is in pH 8.0 solution.100 μ L Enterokinase solution mix with the 2.4mL substrate solution, room temperature (25 ℃), and the variation of fluorescence intensity in the analytical unit time, by the enhancing detection enzymic activity of fluorescence intensity, excitation wavelength 337nm detects wavelength 420nm.Km calculates by the Linewaver-Burk graphing method.

Be convenience of calculation, Enterokinase enzymolysis Gly-Asp-Asp-Asp-Asp-Lys-naphthylamide, reacting 30 seconds fluorescence intensities increases by 1, is defined as 1AU.

In the reaction, reorganization ox Enterokinase consumption is 50nM, and concentration of substrate is respectively 0.05,0.1,0.2,0.4 and 0.8mM.The results are shown in Table 1-4, Fig. 4:

Table 1

	??EKL	??EKLm1	??EKLm2	??EKLm3	??EKLm4	??EKLm5
							??0.05	??0.781	??0.528	??0.465	??0.926	??0.601	??1.279
??0.1	??1.412	??0.949	??0.827	??1.603	??1.075	??2.012
							??0.2	??2.095	??1.523	??1.412	??2.186	??1.639	??2.537
??0.4	??2.667	??2.186	??2.019	??2.904	??2.341	??3.106
							??0.8	??3.004	??2.724	??2.647	??3.271	??2.730	??3.452

Table 2

	??EKLm6	??EKLm7	??EKLm8	??EKLm9	??R&D
						??0.05	??2.579	??0.712	??0.954	??0.525	??0.893
??0.1	??3.251	??1.140	??1.577	??0.983	??1.515
						??0.2	??3.426	??1.685	??2.210	??1.684	??2.164
??0.4	??3.705	??2.374	??2.876	??2.379	??2.784
						??0.8	??3.792	??2.793	??3.365	??2.780	??3.129

Table 3

	??EKL	??EKLm1	??EKLm2	??EKLm3	??EKLm4	??EKLm5
							Regression equation	??Y＝0.0509 ??x+0.2403	??Y＝0.0816 ??x+0.2532	??Y＝0.0949 ??x+0.2528	??Y＝0.0412 ??x+0.2429	??Y＝0.0696x ??+0.2604	??Y＝0.0260 ??x+0.2556
Transverse axis intercept (1/Km) (mM -1)	??-4.7210	??-3.1029	??-2.6639	??-5.8956	??-3.7414	??-9.8308
							??Km(mM)	??0.2118	??0.3223	??0.3754	??0.1696	??0.2673	??0.1017
Vertical axis intercept (1/Vmax) (AU -1)	??0.2403	??0.2532	??0.2528	??0.2429	??0.2604	??0.2556
							??Vmax ??(AU)	??4.1615	??3.9494	??3.9557	??4.1169	??3.8402	??3.9124

Table 4

	??EKLm6	??EKLm7	??EKLm8	??EKLm9	??R&D
						Regression equation	??Y＝0.0065 ??x+0.2539	??Y＝0.0559 ??x+0.2973	??Y＝0.0399 ??x+0.2471	??Y＝0.0836 ??x+0.211	??Y＝0.0429x+0.251 ??9
Transverse axis intercept (1/Km) (mM -1)	??-39.0615	??-5.3184	??-6.1930	??-2.5239	??-5.8718
						??Km(mM)	??0.0256	??0.1880	??0.1615	??0.3962	??0.1703
Vertical axis intercept (1/Vmax) (AU -1)	??0.2539	??0.2973	??0.2471	??0.211	??0.2519
						??Vmax ??(AU)	??3.9386	??3.3636	??4.0469	??4.7393	??3.9698

Conclusion:

The Km of wild-type EKL and Vmax and R﹠amp; The D like product is similar, in 9 mutant, the Km of EKLm3, EKLm5, EKLm6, EKLm7 and EKLm8 compares the decline (aminoacid sequence of EKLm3, EKLm5, EKLm6, EKLm7 and EKLm8 is respectively SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16 through order-checking) that all has in various degree with wild-type, wherein reduce the most significantly with the Km value of EKLm6, promptly EKLm6 has the avidity with substrate the best.

The structure of embodiment 6 pET39b/EKH-EKLm6 fusion protein expression plasmids

Because the Enterpeptidase light chain inclusion body products renaturation difficulty that escherichia expression system obtains, so the yield of activated protein is extremely low.In order to improve the output of EKL activated protein as far as possible, on the basis of adopting the pET39b expression vector, (nucleotide sequence is seen SEQ ID NO.11 through the flexible Linker of one 10 peptide to choose each structural domain of enteropeptidase heavy chain EKH and EKL respectively, aminoacid sequence is seen SEQ ID NO.12) linking to each other constitutes fusion rotein, screen optimized fusion rotein structure by renaturation situation and determination of activity to each fusion rotein, concrete experimental technique is as follows.

To identify that correct pMD18-T-EK is a template, pcr amplification Enteropeptidase heavy chain structural domain 5 and be template amplification light chain coding region dna fragmentation once more with pET39b-EKLm6 (SEQ ID NO:1 and SEQ ID NO:2 have shown nucleotide sequence and the aminoacid sequence of EKLm6 respectively), heavy chain the 5th structural domain upstream primer is 5 '-gaattcggacgacgacgacaagcgtctcttcaatggcacg-3 ', downstream primer is 5 ' cccaccgcctgagcctccaccgccgtagaaacattgtagcag-3 ', light chain upstream of coding region primer is 5-' gaggctcaggcggtgggggttctattgtcggaggaagtgactc-3 ', and the light chain downstream primer is 5 '-aagctttcaatgtagaaaactttgtatcc-3 '.Heavy chain the 5th structural domain upstream primer has EcoRI restriction enzyme site and enteropeptidase recognition site, light chain downstream primer band HindIII restriction enzyme site and terminator codon.Heavy chain the 5th structural domain downstream primer and the overlapping pcr amplification condition of light chain upstream of coding region primer are 94 ℃ of pre-sex change 4min; 45 seconds, 55 ℃ annealing of 94 ℃ of sex change 1min, 72 ℃ of 1min carry out 30 circulations then; Extend 7min at 72 ℃ at last.Pcr amplification product reclaims after agarose gel electrophoresis is identified, with the method connection of overlap PCR, reclaims through the agarose gel electrophoresis purifying, cuts 2hr with HindIII and EcoRI in 37 ℃ of enzymes respectively with the pET39b plasmid DNA.1 * TAE, 0.8% agarose gel electrophoresis reclaims enzyme respectively and cuts product.Insertion fragment after the processing and carrier mixed in 4: 1 in molar ratio, 10 μ L linked systems, and 1 μ l T4DNA ligase enzyme, 16 ℃ of water-baths are spent the night.Connect and press product Transformed E .coli BL21 (DE3) competent cell, select recombinant clone and cultivate, extract plasmid DNA, identify correct recombinant clone through HindIII and EcoRI double digestion, called after pET39b/EKH5-EKLm6.Using the same method, (each structural domain primer of heavy chain is as follows: heavy chain the 1st structural domain upstream primer is 5 '-gaattcggacgacgacgacaagccacctgattcaaggctgtg-3 ' C to structure Enteropeptidase heavy chain structural domain 1,3,4, and downstream primer is 5 ' cccaccgcctgagcctccaccgcc agctgtggcacaagttttattg-3 '; Heavy chain the 3rd structural domain upstream primer is 5 '-gaattcggacgacgacgacaagtgtggagggcctcatgacc-3 ', and downstream primer is 5 ' cccaccgcctgagcctccaccgcc atagccagtagtgaaatttgc-3 '; Heavy chain the 4th structural domain upstream primer is 5 '-gaattcggacgacgacgacaagaaggaagacaattttcagtg-3 ', and downstream primer is 5 ' cccaccgcctgagcctccaccgcc acagtgtgcttcatctgagc-3 '.The light chain primer is the same) with the fusion protein expression plasmid of EKLm6, called after pET39b/EKH1-EKLm6, pET39b/EKH3-EKLm6, pET39b/EKH4-EKLm6 respectively.SEQID NO:3 and SEQ ID NO:4 have shown nucleotide sequence and the aminoacid sequence of EKH1-EKLm6 respectively.SEQ ID NO:5 and SEQ ID NO:6 have shown nucleotide sequence and the aminoacid sequence of EKH3-EKLm6 respectively.SEQ ID NO:7 and SEQ ID NO:8 have shown nucleotide sequence and the aminoacid sequence of EKH4-EKLm6 respectively.SEQ ID NO:9 and SEQ ID NO:10 have shown nucleotide sequence and the aminoacid sequence of EKH5-EKLm6 respectively.

The abduction delivering of embodiment 7 pET39b/EKH-EKLm6 fusion roteins

Method is distinguished abduction delivering EKH1-EKLm6, EKH3-EKLm6, EKH4-EKLm6, EKH5-EKLm6 with embodiment 3.

The renaturation and the purifying of embodiment 8pET39b/EKH-EKLm6 fusion rotein

Method is with embodiment 4, and the result is as shown in table 5, and every 100g EKH5-EKLm6 inclusion body recoverability purifying obtains 113.8mg albumen, and its yield is significantly increased than EKL, EKLm6, EKH1-EKLm6, EKH3-EKLm6 and EKH4-EKLm6.

Table 5

Recombinant protein	Pure protein acquisition amount mg/ hectogram inclusion body
		??EKL	??10.4
??EKLm6	??9.8
		??EKH1-EKLm6	??7.6
??EKH3-EKLm6	??12.6
		??EKH4-EKLm6	??9.5
??EKH5-EKLm6	??113.8

Embodiment 9EKL and the expression of EKLm albumen in pichia spp

Goal gene among pET39b-EKL and the pET39b-EKLm (1-9) is cut with BamHI and EcoRI enzyme, in pPICZ α A (Invitrogen) plasmid of cutting with enzyme of packing into.Connect product and transform CaCl ₂The TOP10F competent cell of method preparation is selected recombinant clone and is cultivated, and extracts plasmid DNA, identifies correct recombinant clone through BamHI and EcoRI double digestion, respectively called after pPICZ α A-EKL and pPICZ α A-EKLm (1-9).The plasmid linearization of Ti Quing subsequently, transformed competence colibacillus yeast cell GS115 (Invitrogen company), the dull and stereotyped preliminary screening of RDB obtains positive recombinant.The activatory engineering bacteria is inoculated in respectively in YPD and the BMGY substratum, puts into shaking table incubated overnight under 30 ℃, 250rpm condition.Seed liquor after the incubated overnight is refunded in the former BMGY substratum, and bannerman's shaking table is cultivated 48h under 30 ℃, 250rpm condition.Add anhydrous methanol and induce, to final concentration be 0.5%.Every 12h sampling, check the enteropeptidase expression, and add anhydrous methanol behind the methanol induction, to final concentration be 0.5%.Receive bottle after inducing 72h, institute's sample thief is analyzed.In full-automatic 30L fermentor tank, exploratory cultivation and fermentation have been carried out simultaneously by the level of loading amount 10L.Engineering bacteria is inoculated in 1L YPD (the divide 2 bottled) substratum, puts into shaking table and under 30 ℃, 250rpm condition, cultivate.When ‰ being 5.0 and microscopy bacterial classification form when qualified, be transferred in the fermentor tank.30 ℃ of culture temperature, ammoniacal liquor is regulated pH5.0, and saturated oxygen gas integration number keeps being not less than 20%.Add 50% glycerine behind the autoclaving according to thalli growth density in the fermenting process.Add anhydrous methanol and induce when cell density reaches OD and is 200～250, control its final concentration and be no more than 1%, this moment, the pH value was controlled at 3.0.Induce the every 12h sampling in back, survey OD and thalline weight in wet base, and measure active.Receive jar after inducing 72h, the centrifuging and taking supernatant is prepared purifying.

EKL that expresses in embodiment 10 pichia spp and the proteic purifying of EKLm

Collect supernatant, through milipore ultra-filtration membrane ultrafiltration and concentration, behind the NaOH accent pH8.0 of concentrated solution with the previous step acquisition, through Chelating Sepharose Fast Flow column purification with 1M.With damping fluid (25mmol/L Tris-HCl pH8.0,0.3M NaCl) flushing balance pillar, with sample on the concentrated solution of pH8.0, flow velocity is 4cm/min, collects to penetrate liquid earlier.Wash to baseline with balance liquid behind the last sample, with wash-out A liquid (25mMTris-HCl pH8.0, the 50mmol/L imidazoles) eluted protein, collect elution peak 1, use wash-out B liquid (25mMTris-HCl pH8.0,200mmol/L imidazoles) eluted protein again, collect elution peak 2, SDS-PAGE detects, and the results are shown in Table 6.

Table 6

Recombinant protein	Pure protein acquisition amount mg/L fermented liquid
		??EKL	??3.7
??EKLm1	??2.5
		??EKLm2	??4.9
??EKLm3	??2.1
		??EKLm4	??3.4
??EKLm5	??4.2
		??EKLm6	??10.3
??EKLm7	??3.8
		??EKLm8	??4.5
??EKLm9	??3.9

Embodiment 11 pET39b/EKLm6, pET39b/EKH-EKLm6, the determination of activity material of pPICZ α A-EKLm6:

Analysis buffer: 50mM Tris, pH 7.5;

Reorganization ox Enterokinase: the present invention's preparation;

Substrate: thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) (Bachem Catalog#M-1300); 5,5 '-dithio-bis (2-nitrobenzoic acid) is (Sigma Catalog#D-8130) (DTNB); 96 hole enzyme plates (Costar, Catalog#92592);

Microplate reader (SpectraMax Plus, Molecular Devices);

Recombination ox intestine kinase 4139-SE, R﹠amp; D company product

Method:

With analysis buffer heavy thin ox Enterokinase is diluted to 0.04 μ g/mL;

With the analysis buffer that contains 200 μ M DTNB Z-Lys-SBzl is diluted to 200 μ M;

Add reorganization ox Enterokinase diluent in 96 orifice plates, 50 μ L/ holes, the blank hole adds analysis buffer, 50 μ L/ holes;

Every hole adds 50 μ L Z-Lys-SBzl/DTNB mixed solutions and begins reaction;

Read A405nm with kinetics model, totally 5 minutes;

Calculate reorganization ox Enterokinase specific activity:

Enzyme and substrate final concentration (amount) in 96 orifice plates

Reorganization ox Enterokinase:2ng

Recombination ox intestine kinase 4139-SE:2ng

DTNB：100μM

Substrate: Z-Lys-SBz:100 μ M

A405/min=(V _EnzymeThe blank max of max-V) (mOD/min)/correction factor cm/1000

Active (nmole/min/ μ g)={ [A405/min (calculating) * volume (0.0001L)]/[optical extinction coefficient (13260M by following formula ^-1Cm ^-1) * enzyme amount (μ g)] } * 10 ⁹

The results are shown in Table 7:

Table 7

	??EKL ??(E.coli)	??EKLm6 ??(E.coli)	??EKH5-EKLm6 ??(E.coli)	??EKLm6 ??(yeast)	Recombination ox intestine kinase 4139-SE
						V enzyme max	??14	??119	??102	??241	??13
??V Blankmax	??2	??7	??6	??8	??2

??V EnzymeThe blank max of max-V	??12	??112	??96	??233	??11
						Active	??45.2	??422.3	??362.0	??878.6	??41.5

Conclusion:

Wild-type EKL activity and R﹠amp; The D like product is similar, and EKLm6, EKH5-EKLm6 activity that colibacillus is expressed have improved about 10 times than wild-type, and the EKLm6 activity of yeast expression is 2 times of same protein of escherichia coli expression.Embodiment 12: temperature of reaction is cut the influence experiment of thioredoxin fusion rotein thioredoxin-human interleukin-11 (Trx/hIL-11) to enzyme

The fusion rotein thioredoxin-human interleukin-11 (Trx/hIL-11) (the following Trx/hIL-11 that all claims) that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 2ng EKH5-EKLm6 reaction, reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, enzyme Qie Wendu are respectively 10 ℃, 20 ℃ and 30 ℃, reaction 20hr.Reaction product utilizes SDS-PAGE to detect.

SDS-PAGE result shows that EKH5-EKLm6 has best enzyme at 20 ℃ and cuts the vigor (see figure 5)

Embodiment 13: the reaction times is cut the influence experiment of thioredoxin fusion rotein Trx/hIL-11 to enzyme

The fusion rotein thioredoxin-human interleukin-11 (Trx/hIL-11) that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 2ng EKH5-EKLm6 reaction, and reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, temperature of reaction is 20 ℃, the reaction times is respectively 5hr, 10hr, 20hr and 40hr.Reaction product utilizes SDS-PAGE to detect.

SDS-PAGE result shows that EKH5-EKLm6 cuts Trx/hIL-11 at 20 ℃ of enzymes, but the 20hr enzyme cuts full (see figure 6).

Embodiment 14: enzyme is cut the optimal proportions of thioredoxin fusion rotein Trx/hIL-11

The fusion rotein thioredoxin-human interleukin-11 (Trx/hIL-11) that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 0,0.02,0.04,0.08,0.16,0.32,0.64 and 1.28ng EKH5-EKLm6 reaction, reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, 20 ℃ * 20hr of reaction conditions.Reaction product utilizes SDS-PAGE to detect.

SDS-PAGE result shows that EKH5-EKLm6 cuts Trx/hIL-11 at 20 ℃ of enzymes, reaction 20hr, and at enzyme: (m: m) (mass ratio is as follows) can cut full (see figure 7) by enzyme to substrate=1: 20000.

The Detection of Stability of embodiment 15:EKH5-EKLm6

The unfavourable condition that the EKH5-EKLm6 simulation may meet with in actual preservation process then detects the ability that its enzyme is cut Trx/hIL-11, and with 20 μ g and 1.28ng EKH5-EKLm6 reaction, reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, 20 ℃ * 20hr of reaction conditions.Reaction product utilizes SDS-PAGE to detect.Intend the investigation that keeps sample under the preservation condition :-20 ℃, preserved 6 months;

Multigelation :-20 ℃ and 37 ℃ 3 times repeatedly;

High temperature: placed 1,3,5,7,10 day for 37 ℃.

SDS-PAGE result shows that EKH5-EKLm6 cuts activity to enzyme and has no significant effect (see figure 8) under above-mentioned preservation condition.

Experimental example 16 temperature of reaction are cut the influence experiment of thioredoxin fusion rotein Trx-MBL-CLR to enzyme

The fusion rotein Trx-MBL-CLR that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 2ng EKH5-EKLm6 reaction, and reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂And 0.1%Tween 20, reaction volume 20 μ L, enzyme Qie Wendu are respectively 10 ℃, 20 ℃ and 30 ℃, reaction 20hr.Reaction product utilizes SDS-PAGE to detect.The results are shown in Figure 11SDS-PAGE result and show, EKH5-EKLm6 has best enzyme at 20 ℃ and cuts vigor.

17 reaction times of embodiment are cut the influence experiment of thioredoxin fusion rotein Trx-MBL-CLR to enzyme

The fusion rotein Trx-MBL-CLR that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 2ngEKH5-EKLm6 reaction, and reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, temperature of reaction is 20 ℃, the reaction times is respectively 5hr, 10hr, 20hr and 40hr.Reaction product utilizes SDS-PAGE to detect.The results are shown in Figure 12

SDS-PAGE result shows that EKH5-EKLm6 cuts Trx-MBL-CLR at 20 ℃ of enzymes, but the 20hr enzyme cuts entirely.

Embodiment 18 enzymes are cut the optimal proportions experiment of thioredoxin fusion rotein Trx-MBL-CLR

The fusion rotein Trx-MBL-CLR that contains enteropeptidase restriction enzyme site (DDDDK) is with 20 μ g and 0,0.02,0.04,0.08, and 0.16,0.32,0.64 and the 1.28ngEKH5-EKLm6 reaction, reaction system is 0.1M Tris-HCl (pH 8.0), 1mMCaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, 20 ℃ * 20hr of reaction conditions.Reaction product utilizes SDS-PAGE to detect.The results are shown in Figure 13

SDS-PAGE result shows that EKH5-EKLm6 cuts Trx-MBL-CLR at 20 ℃ of enzymes, and reaction 20hr is at enzyme: substrate=1: 20000 (m: m) can cut entirely by enzyme.

The Detection of Stability of embodiment 19EKH5-EKLm6

The unfavourable condition that the EKH5-EKLm6 simulation may meet with in actual preservation process then detects the ability that its enzyme is cut Trx-MBL-CLR, and with 20 μ g and 1.28ng EKH5-EKLm6 reaction, reaction system is 0.1M Tris-HCl (pH 8.0), 1mM CaCl ₂, and 0.1%Tween 20, reaction volume 20 μ L, 20 ℃ * 20hr of reaction conditions.Reaction product utilizes SDS-PAGE to detect.Intend the investigation that keeps sample under the preservation condition :-20 ℃, preserved 6 months;

Multigelation :-20 ℃ and 37 ℃ 3 times repeatedly;

High temperature: placed 1,3,5,7,10 day for 37 ℃.

SDS-PAGE the results are shown in Figure 14, and the result shows that EKH5-EKLm6 cuts activity to enzyme and has no significant effect under above-mentioned preservation condition.

Sequence table

SEQUENCE?LISTING

＜110〉Shanghai Zhangjiang Biological Technology Co

＜120〉recombination ox intestine kinase, its preparation method and application

<130>CN0111370.3

<150>200710040551.0

<151>2007-05-11

<150>200710040552.5

<151>2007-05-11

<160>16

<170>PatentIn?version?3.4

<210>1

<211>705

<212>DNA

<213>Unknown

<220>

＜223〉light chain mutant nucleotide sequence

<400>1

attgtcggag?gaagtgactc?cagagaagga?gcctggcctt?gggtcgttgc?tctgtatttc??????60

gacgatcaac?aggtctgcgg?agcttctctg?gtgagcaggg?attggctggt?gtcggccgcc?????120

cactgcgtgt?acgggagaaa?tatggagccg?tctaagtgga?aagcagtgct?aggcctgcat?????180

atggcatcaa?atctgacttc?tcctcagata?gaaactaggt?tgattgacca?aattgtcata?????240

aacccacact?acaataaacg?gagaaagaac?aatgacattg?ccatgatgca?tcttgaaatg?????300

aaagtgaact?acacagatta?tatacagcct?atttgtttac?cagaagaaaa?tcaagttttt?????360

cccccaggaa?gaatttgttc?tattgctggc?tggggggcac?ttatatatca?aggttctact?????420

gcagacgtac?tgcaagaagc?tgacgttccc?cttctatcaa?atgagaaatg?tcaacaacag?????480

atgccagaac?gtaacattac?ggaaaatatg?gtgtgtgcag?gctatgaagc?aggaggggta?????540

gattcttgtc?agggggattc?aggcggacca?ctcatgtgcc?aagaaaacaa?cagatggctc?????600

ctggctggcg?tgacgtcatt?tggacgtcaa?tgtgcactgc?ctaatcgccc?aggggtgtat?????660

gcccgggtcc?caaggttcac?agagtggata?caaagttttc?tacat?????????????????????705

<210>2

<211>235

<212>PRT

<213>unknown

<220>

＜223〉light chain mutant aminoacid sequence

<400>2

Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val

1???????????????5???????????????????10??????????????????15

Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser

20??????????????????25??????????????????30

Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met

35??????????????????40??????????????????45

Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn

50??????????????????55??????????????????60

Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile

65??????????????????70??????????????????75??????????????????80

Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met

85??????????????????90??????????????????95

His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys

100?????????????????105?????????????????110

Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile

115?????????????????120?????????????????125

Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140

Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln

145?????????????????150?????????????????155?????????????????160

Met?Pro?Glu?Arg?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu

165?????????????????170?????????????????175

Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met

180?????????????????185?????????????????190

Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Phe?Gly

195?????????????????200?????????????????205

Arg?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro

210?????????????????215??????????????????220

Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

225?????????????????230?????????????????235

<210>3

<211>855

<212>DNA

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH1-EKLm6 nucleotide sequence

<400>3

ccacctgatt?caaggctgtg?tgctgatgct?ctaaagtgca?tagcaattga?tttattttgt?????60

gatggagaat?taaactgtcc?agatggctct?gatgaagaca?ataaaacttg?tgccacagct????120

ggcggtggag?gctcaggcgg?tgggggttct?attgtcggag?gaagtgactc?cagagaagga????180

gcctggcctt?gggtcgttgc?tctgtatttc?gacgatcaac?aggtctgcgg?agcttctctg????240

gtgagcaggg?attggctggt?gtcggccgcc?cactgcgtgt?acgggagaaa?tatggagccg????300

tctaagtgga?aagcagtgct?aggcctgcat?atggcatcaa?atctgacttc?tcctcagata????360

gaaactaggt?tgattgacca?aattgtcata?aacccacact?acaataaacg?gagaaagaac????420

aatgacattg?ccatgatgca?tcttgaaatg?aaagtgaact?acacagatta?tatacagcct????480

atttgtttac?cagaagaaaa?tcaagttttt?cccccaggaa?gaatttgttc?tattgctggc????540

tggggggcac?ttatatatca?aggttctact?gcagacgtac?tgcaagaagc?tgacgttccc????600

cttctatcaa?atgagaaatg?tcaacaacag?atgccagaac?gtaacattac?ggaaaatatg????660

gtgtgtgcag?gctatgaagc?aggaggggta?gattcttgtc?agggggattc?aggcggacca????720

ctcatgtgcc?aagaaaacaa?cagatggctc?ctggctggcg?tgacgtcatt?tggacgtcaa????780

tgtgcactgc?ctaatcgccc?aggggtgtat?gcccgggtcc?caaggttcac?agagtggata????840

caaagttttc?tacat?????????????????????????????????????????????????????855

<210>4

<211>285

<212>PRT

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH1-EKLm6 aminoacid sequence

<400>4

Pro?Pro?Asp?Ser?Arg?Leu?Cys?Ala?Asp?Ala?Leu?Lys?Cys?Ile?Ala?Ile

1???????????????5???????????????????10??????????????????15

Asp?Leu?Phe?Cys?Asp?Gly?Glu?Leu?Asn?Cys?Pro?Asp?Gly?Ser?Asp?Glu

20??????????????????25??????????????????30

Asp?Asn?Lys?Thr?Cys?Ala?Thr?Ala?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly

35??????????????????40??????????????????45

Gly?Ser?Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp

50??????????????????55??????????????????60

Val?Val?Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu

65??????????????????70??????????????????75??????????????????80

Val?Ser?Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg

85??????????????????90??????????????????95

Asn?Met?Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala

100?????????????????105?????????????????110

Ser?Asn?Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile

115?????????????????120?????????????????125

Val?Ile?Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala

130?????????????????135?????????????????140

Met?Met?His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro

145?????????????????150?????????????????155?????????????????160

Ile?Cys?Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys

165?????????????????170?????????????????175

Ser?Ile?Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp

180?????????????????185?????????????????190

Val?Leu?Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln

195?????????????????200?????????????????205

Gln?Gln?Met?Pro?Glu?Arg?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly

210?????????????????215?????????????????220

Tyr?Glu?Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro

225?????????????????230?????????????????235?????????????????240

Leu?Met?Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser

245?????????????????250?????????????????255

Phe?Gly?Arg?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg

260?????????????????265?????????????????270

Val?Pro?Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

275?????????????????280?????????????????285

<210>5

<211>1071

<212>DNA

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH3-EKLm6 gene order

<400>5

tgtggagggc?ctcatgacct?gtgggagcca?aatacaacat?tcacgtctat?aaacttccca???60

aacagctacc?ctaatcaggc?tttctgtatt?tggaatttaa?atgcacaaaa?gggaaaaaat??120

attcagctcc?actttcaaga?atttgacctg?gaaaatattg?cagatgtagt?tgaaatcaga??180

gatggtgaag?gagatgattc?cttgttctta?gctgtgtaca?caggccctgg?tccagtaaac??240

gatgtgttct?caaccaccaa?ccgaatgact?gtgcttttta?tcactgataa?tatgctggca??300

aaacagggat?ttaaagcaaa?tttcactact?ggctatggcg?gtggaggctc?aggcggtggg??360

ggttctattg?tcggaggaag?tgactccaga?gaaggagcct?ggccttgggt?cgttgctctg??420

tatttcgacg?atcaacaggt?ctgcggagct?tctctggtga?gcagggattg?gctggtgtcg??480

gccgcccact?gcgtgtacgg?gagaaatatg?gagccgtcta?agtggaaagc?agtgctaggc??540

ctgcatatgg?catcaaatct?gacttctcct?cagatagaaa?ctaggttgat?tgaccaaatt??600

gtcataaacc?cacactacaa?taaacggaga?aagaacaatg?acattgccat?gatgcatctt????660

gaaatgaaag?tgaactacac?agattatata?cagcctattt?gtttaccaga?agaaaatcaa????720

gtttttcccc?caggaagaat?ttgttctatt?gctggctggg?gggcacttat?atatcaaggt????780

tctactgcag?acgtactgca?agaagctgac?gttccccttc?tatcaaatga?gaaatgtcaa????840

caacagatgc?cagaacgtaa?cattacggaa?aatatggtgt?gtgcaggcta?tgaagcagga????900

ggggtagatt?cttgtcaggg?ggattcaggc?ggaccactca?tgtgccaaga?aaacaaoaga????960

tggctcctgg?ctggcgtgac?gtcatttgga?cgtcaatgtg?cactgcctaa?tcgcccaggg???1020

gtgtatgccc?gggtcccaag?gttcacagag?tggatacaaa?gttttctaca?t????????????1071

<210>6

<211>357

<212>PRT

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH3-EKLm6 aminoacid sequence

<400>6

Cys?Gly?Gly?Pro?His?Asp?Leu?Trp?Glu?Pro?Asn?Thr?Thr?Phe?Thr?Ser

1???????????????5???????????????????10??????????????????15

Ile?Asn?Phe?Pro?Asn?Ser?Tyr?Pro?Asn?Gln?Ala?Phe?Cys?Ile?Trp?Asn

20??????????????????25??????????????????30

Leu?Asn?Ala?Gln?Lys?Gly?Lys?Asn?Ile?Gln?Leu?His?Phe?Gln?Glu?Phe

35??????????????????40??????????????????45

Asp?Leu?Glu?Asn?Ile?Ala?Asp?Val?Val?Glu?Ile?Arg?Asp?Gly?Glu?Gly

50??????????????????55??????????????????60

Asp?Asp?Ser?Leu?Phe?Leu?Ala?Val?Tyr?Thr?Gly?Pro?Gly?Pro?Val?Asn

65??????????????????70??????????????????75??????????????????80

Asp?Val?Phe?Ser?Thr?Thr?Asn?Arg?Met?Thr?Val?Leu?Phe?Ile?Thr?Asp

85??????????????????90??????????????????95

Asn?Met?Leu?Ala?Lys?Gln?Gly?Phe?Lys?Ala?Asn?Phe?Thr?Thr?Gly?Tyr

100?????????????????105?????????????????110

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ile?Val?Gly?Gly?Ser?Asp

115?????????????????120?????????????????125

Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val?Ala?Leu?Tyr?Phe?Asp?Asp

130?????????????????135?????????????????140

Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser?Arg?Asp?Trp?Leu?Val?Ser

145?????????????????150?????????????????155?????????????????160

Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met?Glu?Pro?Ser?Lys?Trp?Lys

165?????????????????170?????????????????175

Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn?Leu?Thr?Ser?Pro?Gln?Ile

180?????????????????185?????????????????190

Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile?Asn?Pro?His?Tyr?Asn?Lys

195?????????????????200?????????????????205

Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met?His?Leu?Glu?Met?Lys?Val

210?????????????????215?????????????????220

Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys?Leu?Pro?Glu?Glu?Asn?Gln

225?????????????????230?????????????????235?????????????????240

Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile?Ala?Gly?Trp?Gly?Ala?Leu

245?????????????????250?????????????????255

Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu?Gln?Glu?Ala?Asp?Val?Pro

260?????????????????265?????????????????270

Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln?Met?Pro?Glu?Arg?Asn?Ile

275?????????????????280?????????????????285

Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu?Ala?Gly?Gly?Val?Asp?Ser

290?????????????????295?????????????????300

Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met?Cys?Gln?Glu?Asn?Asn?Arg

305?????????????????310?????????????????315?????????????????320

Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Phe?Gly?Arg?Gln?Cys?Ala?Leu?Pro

325?????????????????330?????????????????335

Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro?Arg?Phe?Thr?Glu?Trp?Ile

340?????????????????345?????????????????350

Gln?Ser?Phe?Leu?His

355

<210>7

<211>837

<212>DNA

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH4-EKLm6 gene order

<400>7

aaggaagaca?attttcagtg?caaggatggg?gagtgtattc?cgctggtgaa?tctctgtgac?????60

ggttttccac?actgtaagga?tggctcagat?gaagcacact?gtggcggtgg?aggctcaggc????120

ggtgggggtt?ctattgtcgg?aggaagtgac?tccagagaag?gagcctggcc?ttgggtcgtt????180

gctctgtatt?tcgacgatca?acaggtctgc?ggagcttctc?tggtgagcag?ggattggctg????240

gtgtcggccg?cccactgcgt?gtacgggaga?aatatggagc?cgtctaagtg?gaaagcagtg????300

ctaggcctgc?atatggcatc?aaatctgact?tctcctcaga?tagaaactag?gttgattgac????360

caaattgtca?taaacccaca?ctacaataaa?cggagaaaga?acaatgacat?tgccatgatg????420

catcttgaaa?tgaaagtgaa?ctacacagat?tatatacagc?ctatttgttt?accagaagaa????480

aatcaagttt?ttcccccagg?aagaatttgt?tctattgctg?gctggggggc?acttatatat????540

caaggttcta?ctgcagacgt?actgcaagaa?gctgacgttc?cccttctatc?aaatgagaaa????600

tgtcaacaac?agatgccaga?acgtaacatt?acggaaaata?tggtgtgtgc?aggctatgaa????660

gcaggagggg?tagattcttg?tcagggggat?tcaggcggac?cactcatgtg?ccaagaaaac????720

aacagatggc?tcctggctgg?cgtgacgtca?tttggacgtc?aatgtgcact?gcctaatcgc????780

ccaggggtgt?atgcccgggt?cccaaggttc?acagagtgga?tacaaagttt?tctacat???????837

<210>8

<211>279

<212>PRT

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH4-EKLm6 aminoacid sequence

<400>8

Lys?Glu?Asp?Asn?Phe?Gln?Cys?Lys?Asp?Gly?Glu?Cys?Ile?Pro?Leu?Val

1???????????????5???????????????????10??????????????????15

Asn?Leu?Cys?Asp?Gly?Phe?Pro?His?Cys?Lys?Asp?Gly?Ser?Asp?Glu?Ala

20??????????????????25??????????????????30

His?Cys?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Ile?Val?Gly?Gly

35??????????????????40??????????????????45

Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val?Ala?Leu?Tyr?Phe

50??????????????????55??????????????????60

Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser?Arg?Asp?Trp?Leu

65??????????????????70??????????????????75??????????????????80

Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met?Glu?Pro?Ser?Lys

85??????????????????90??????????????????95

Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn?Leu?Thr?Ser?Pro

100?????????????????105?????????????????110

Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile?Asn?Pro?His?Tyr

115?????????????????120?????????????????125

Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met?His?Leu?Glu?Met

130?????????????????135?????????????????140

Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys?Leu?Pro?Glu?Glu

145?????????????????150?????????????????155?????????????????160

Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile?Ala?Gly?Trp?Gly

165?????????????????170?????????????????175

Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu?Gln?Glu?Ala?Asp

180?????????????????185?????????????????190

Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln?Met?Pro?Glu?Arg

195?????????????????200?????????????????205

Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu?Ala?Gly?Gly?Val

210?????????????????215?????????????????220

Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met?Cys?Gln?Glu?Asn

225?????????????????230?????????????????235?????????????????240

Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Phe?Gly?Arg?Gln?Cys?Ala

245?????????????????250?????????????????255

Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro?Arg?Phe?Thr?Glu

260?????????????????265?????????????????270

Trp?Ile?Gln?Ser?Phe?Leu?His

275

<210>9

<211>1008

<212>DNA

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH5-EKLm6 gene order

<400>9

cgtctcttca?atggcacgac?agacagcagt?ggtttggtgc?agttcaggat?ccaaagcata???60

tggcatgtag?cctgtgccga?gaactggaca?acccagatct?cagatgatgt?gtgtcagctg??120

ctgggactag?ggactggaaa?ctcatccgtg?ccaacctttt?ctactggagg?tggaccatat??180

gtaaatttaa?acacagcacc?taatggcagc?ttaatactaa?cgccaagcca?acagtgctta??240

gaggattcac?tgattctgct?acaatgtaac?tacggcggtg?gaggctcagg?cggtgggggt??300

tctattgtcg?gaggaagtga?ctccagagaa?ggagcctggc?cttgggtcgt?tgctctgtat??360

ttcgacgatc?aacaggtctg?cggagcttct?ctggtgagca?gggattggct?ggtgtcggcc??420

gcccactgcg?tgtacgggag?aaatatggag?ccgtctaagt?ggaaagcagt?gctaggcctg??480

catatggcat?caaatctgac?ttctcctcag?atagaaacta?ggttgattga?ccaaattgtc??540

ataaacccac?actacaataa?acggagaaag?aacaatgaca?ttgccatgat?gcatcttgaa??600

atgaaagtga?actacacaga?ttatatacag?cctatttgtt?taccagaaga?aaatcaagtt??660

tttcccccag?gaagaatttg?ttctattgct?ggctgggggg?cacttatata?tcaaggttct??720

actgcagacg?tactgcaaga?agctgacgtt?ccccttctat?caaatgagaa?atgtcaacaa??780

cagatgccag?aacgtaacat?tacggaaaat?atggtgtgtg?caggctatga?agcaggaggg??840

gtagattctt?gtcaggggga?ttcaggcgga?ccactcatgt?gccaagaaaa?caacagatgg??900

ctcctggctg?gcgtgacgtc?atttggacgt?caatgtgcac?tgcctaatcg?cccaggggtg??960

tatgcccggg?tcccaaggtt?cacagagtgg?atacaaagtt?ttctacat??????????????1008

<210>10

<211>336

<212>PRT

<213>Unknown

<220>

＜223〉recombination ox intestine kinase heavy chain structural domain-light chain mutant fusion protein EKH5-EKLm6 aminoacid sequence

<400>10

Arg?Leu?Phe?Asn?Gly?Thr?Thr?Asp?Ser?Ser?Gly?Leu?Val?Gln?Phe?Arg

1???????????????5???????????????????10??????????????????15

Ile?Gln?Ser?Ile?Trp?His?Val?Ala?Cys?Ala?Glu?Asn?Trp?Thr?Thr?Gln

20??????????????????25??????????????????30

Ile?Ser?Asp?Asp?Val?Cys?Gln?Leu?Leu?Gly?Leu?Gly?Thr?Gly?Asn?Ser

35??????????????????40??????????????????45

Ser?Val?Pro?Thr?Phe?Ser?Thr?Gly?Gly?Gly?Pro?Tyr?Val?Asn?Leu?Asn

50??????????????????55??????????????????60

Thr?Ala?Pro?Asn?Gly?Ser?Leu?Ile?Leu?Thr?Pro?Ser?Gln?Gln?Cys?Leu

65??????????????????70??????????????????75??????????????????80

Glu?Asp?Ser?Leu?Ile?Leu?Leu?Gln?Cys?Asn?Tyr?Gly?Gly?Gly?Gly?Ser

85??????????????????90??????????????????95

Gly?Gly?Gly?Gly?Ser?Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala

100?????????????????105?????????????????110

Trp?Pro?Trp?Val?Val?Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly

115?????????????????120?????????????????125

Ala?Ser?Leu?Val?Ser?Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val

130?????????????????135?????????????????140

Tyr?Gly?Arg?Asn?Met?Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu

145?????????????????150?????????????????155?????????????????160

His?Met?Ala?Ser?Asn?Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile

165?????????????????170?????????????????175

Asp?Gln?Ile?Val?Ile?Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn

180?????????????????185?????????????????190

Asp?Ile?Ala?Met?Met?His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr

195?????????????????200?????????????????205

Ile?Gln?Pro?Ile?Cys?Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly

210?????????????????215?????????????????220

Arg?Ile?Cys?Ser?Ile?Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser

225?????????????????230?????????????????235?????????????????240

Thr?Ala?Asp?Val?Leu?Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu

245?????????????????250?????????????????255

Lys?Cys?Gln?Gln?Gln?Met?Pro?Glu?Arg?Asn?Ile?Thr?Glu?Asn?Met?Val

260?????????????????265?????????????????270

Cys?Ala?Gly?Tyr?Glu?Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser

275?????????????????280?????????????????285

Gly?Gly?Pro?Leu?Met?Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly

290?????????????????295?????????????????300

Val?Thr?Ser?Phe?Gly?Arg?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val

305?????????????????310?????????????????315?????????????????320

Tyr?Ala?Arg?Val?Pro?Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

325?????????????????330?????????????????335

<210>11

<211>30

<212>DNA

<213>Unknown

<220>

＜223〉LINKER nucleotide sequence

<400>11

ggcggtggag?gctcaggcgg?tgggggttct???????????????????????????????????????30

<210>12

<211>10

<212>PRT

<213>Unknown

<220>

＜223〉LINKER aminoacid sequence

<400>12

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser

1???????????????5???????????????????10

<210>13

<211>235

<212>PRT

<213>Unknown

<220>

＜223〉Enteropeptidase light chain mutant EKLm3 aminoacid sequence

<400>13

Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val

1???????????????5???????????????????10??????????????????15

Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser

20??????????????????25??????????????????30

Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met

35??????????????????40??????????????????45

Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn

50??????????????????55??????????????????60

Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile

65??????????????????70??????????????????75??????????????????80

Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met

85??????????????????90??????????????????95

His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys

100?????????????????105?????????????????110

Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile

115?????????????????120?????????????????125

Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140

Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln

145?????????????????150?????????????????155?????????????????160

Met?Pro?Glu?Tyr?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu

165?????????????????170?????????????????175

Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met

180?????????????????185?????????????????190

Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Phe?Gly

195?????????????????200?????????????????205

Arg?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro

210?????????????????215?????????????????220

Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

225?????????????????230?????????????????235

<210>14

<211>235

<212>PRT

<213>Unknown

<220>

＜223〉Enteropeptidase light chain mutant EKLm5 aminoacid sequence

<400>14

Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val

1???????????????5???????????????????10??????????????????15

Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser

20??????????????????25??????????????????30

Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met

35??????????????????40??????????????????45

Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn

50??????????????????55??????????????????60

Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile

65??????????????????70??????????????????75??????????????????80

Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met

85??????????????????90??????????????????95

His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys

100?????????????????105?????????????????110

Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile

115?????????????????120?????????????????125

Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140

Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln

145?????????????????150?????????????????155?????????????????160

Met?Pro?Glu?Tyr?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu

165?????????????????170?????????????????175

Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met

180?????????????????185?????????????????190

Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Arg?Gly

195?????????????????200?????????????????205

Tyr?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro

210?????????????????215?????????????????220

Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

225?????????????????230?????????????????235

<210>15

<211>235

<212>PRT

<213>Unknown

<220>

＜223〉Enteropeptidase light chain mutant EKLm7 aminoacid sequence

<400>15

Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val

1???????????????5???????????????????10??????????????????15

Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser

20??????????????????25??????????????????30

Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met

35??????????????????40??????????????????45

Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn

50??????????????????55??????????????????60

Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile

65??????????????????70??????????????????75??????????????????80

Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met

85??????????????????90??????????????????95

His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys

100?????????????????105?????????????????110

Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile

115?????????????????120?????????????????125

Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140

Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln

145?????????????????150?????????????????155?????????????????160

Met?Pro?Glu?Tyr?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu

165?????????????????170?????????????????175

Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met

180?????????????????185?????????????????190

Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Phe?Gly

195?????????????????200?????????????????205

Tyr?Arg?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro

210?????????????????215?????????????????220

Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

225?????????????????230?????????????????235

<210>16

<211>235

<212>PRT

<213>Unknown

<220>

＜223〉Enteropeptidase light chain mutant EKLm8 aminoacid sequence

<400>16

Ile?Val?Gly?Gly?Ser?Asp?Ser?Arg?Glu?Gly?Ala?Trp?Pro?Trp?Val?Val

1???????????????5???????????????????10??????????????????15

Ala?Leu?Tyr?Phe?Asp?Asp?Gln?Gln?Val?Cys?Gly?Ala?Ser?Leu?Val?Ser

20??????????????????25??????????????????30

Arg?Asp?Trp?Leu?Val?Ser?Ala?Ala?His?Cys?Val?Tyr?Gly?Arg?Asn?Met

35??????????????????40??????????????????45

Glu?Pro?Ser?Lys?Trp?Lys?Ala?Val?Leu?Gly?Leu?His?Met?Ala?Ser?Asn

50?????????????????55???????????????????60

Leu?Thr?Ser?Pro?Gln?Ile?Glu?Thr?Arg?Leu?Ile?Asp?Gln?Ile?Val?Ile

65??????????????????70??????????????????75??????????????????80

Asn?Pro?His?Tyr?Asn?Lys?Arg?Arg?Lys?Asn?Asn?Asp?Ile?Ala?Met?Met

85??????????????????90??????????????????95

His?Leu?Glu?Met?Lys?Val?Asn?Tyr?Thr?Asp?Tyr?Ile?Gln?Pro?Ile?Cys

100?????????????????105?????????????????110

Leu?Pro?Glu?Glu?Asn?Gln?Val?Phe?Pro?Pro?Gly?Arg?Ile?Cys?Ser?Ile

115?????????????????120?????????????????125

Ala?Gly?Trp?Gly?Ala?Leu?Ile?Tyr?Gln?Gly?Ser?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140

Gln?Glu?Ala?Asp?Val?Pro?Leu?Leu?Ser?Asn?Glu?Lys?Cys?Gln?Gln?Gln

145?????????????????150?????????????????155?????????????????160

Met?Pro?Glu?Tyr?Asn?Ile?Thr?Glu?Asn?Met?Val?Cys?Ala?Gly?Tyr?Glu

165?????????????????170?????????????????175

Ala?Gly?Gly?Val?Asp?Ser?Cys?Gln?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Met

180?????????????????185?????????????????190

Cys?Gln?Glu?Asn?Asn?Arg?Trp?Leu?Leu?Ala?Gly?Val?Thr?Ser?Arg?Gly

195?????????????????200?????????????????205

Arg?Gln?Cys?Ala?Leu?Pro?Asn?Arg?Pro?Gly?Val?Tyr?Ala?Arg?Val?Pro

210?????????????????215?????????????????220

Arg?Phe?Thr?Glu?Trp?Ile?Gln?Ser?Phe?Leu?His

225?????????????????230?????????????????235

Claims (1)

1. recombination ox intestine kinase light chain mutant, its aminoacid sequence is SEQ ID NO:14.

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