CN101092598A - Using methanol yeast to produce human kallikrein - 1 - Google Patents

Abstract

This invention relates to a method for producing recombinant human kallikrein-1 (r-HK1) with Pichia pastoris. This invention also describes molecular weight, isoelectric point, and sugar content and glycosyl modification of r-HK1. It is proven by in vivo and in vitro activity experiments that r-HK1 can be used to treat and prevent cerebral infraction.

Description

Using methanol yeast to produce human kallikrein-1-1

One, technical field

The present invention adopts engineered means; obtain a kind of novel recombinant protein on a large scale by methanol yeast (Pichia pastoris) expression system--recombinant human kallikrein-1 (Recombinant HumanKallikrein-1, r-hK that-----methanol yeast is expressed ₁), especially to r-hK ₁The research of molecular property, and body is interior, the mensuration and the test of external activity, shows r-hK ₁Be expected to become the medicine of treatment cerebral infarction.

Two, background technology

As far back as nineteen twenty-six, Frey etc. have just obtained a kind of composition that can cause blood pressure drops from human urine, be referred to as kallidinogenase at that time, but, investigator afterwards finds, this protease composition also is distributed widely among blood plasma, pancreas, kidney, sialisterium, intestines, the pancreatic juice, and especially content is the highest in the pancreas.Nineteen thirty, the Kraut H. of Greece etc. will be from this composition called after Kallikrein (Greek Kallikreas is ' pancreas ', so claim that the protease that obtains in the pancreas is Kallikrein) of pancreas separation and Extraction.But along with the further investigation to aspects such as this proteinoid enzyme function, distribution and the mechanisms of action, at different times, the researchist has different appellations to Kallikrein, for example Kininogenase, Kallidinogenase and Kininogenin.In fact, in Chinese translation, ' kallikrein ' ' kininogenase ' and appellations such as ' kininases ' are also used with.At present, the use of domestic Chinese translation to Kallikrein is disunity also, even in the domestic in recent years article of delivering still to using different titles with a kind of composition, for example ' urinary kallidinogenase ', ' UK ', ' Pancreatic Kininogenase ', ' pancreas kallikrein ', ' tissue kallikrein ' etc.

As if Kallikrein one speech is translated into ' kallikrein ' in this patent, includes the root of prokinin or kassinin kinin one speech in these three speech of Kininogenase, Kallidinogenase and Kininogenin, and it is more appropriate to be called ' kininogenase '.Though ' kallikrein ' or ' kininogenase ' found in particular organization's organs such as pancreas the earliest, but, because it distributes extensively in animal body, kind is many, so, in the description of this patent, will no longer limit certain kallikrein of mentioning with words such as tissue or organ or sources.

In early days, the investigator is divided into blood plasma type and tissue-type two big classes with people's kallikrein (Human Kallikrein, hereafter hK).Blood plasma type hK expresses in people's liver, and secretion enters blood again, and its participates in physiological processs such as the adjusting of the solidifying of blood, fibrinous hydrolysis, blood pressure and inflammatory reaction; And tissue-type hK, be to produce at positions such as pancreas, kidney, sialisterium, mammary gland, ovary, testis and prostate glands, can to some polypeptide (as, prokinin, proinsulin, feritin are former etc.) translate post-treatment, therefrom discharge and have the physiologically active peptide class (as, kassinin kinin, Regular Insulin, feritin etc.).Now, according to the height of content and the sequencing of discovery, all the tissue-type hK except that blood plasma type hK are named in unification in the world, are followed successively by hK ₁, hK ₂, hK ₃... .hK ₁₅Deng, have been found that at present and name (Chen Yu spring foreign medical science physiology, pathology science and clinical fascicle .2003,23 (4): 386～88 are arranged 15 kinds more than; Yousef GM, ObiezuCV, etal Human tissue kallikreins:from gene structure to function and clinical applications.Adv.in Clin.Chem.2005,39:11-79.).Review the described tissue-type hK of early stage investigator, just be not difficult to find that it does not comprise the tissue-type hK of these kinds of finding afterwards and naming from hK in pancreas, kidney, sialisterium and the urine.In fact, said tissue-type kallikrein, pancreas kallikrein, sialisterium kallikrein etc. all are with a kind of composition by new criteria for classification in the document in one's early years, should be referred to as to be people's kallikrein-1 (Human Kallikrein 1, abbreviation hK ₁), this also is the object that this patent will be described.In this patent with the hK of yeast secreted expression ₁(Recombinant Human kallikrein-1 is called for short r-hK to be called the recombinant human kallikrein-1 ₁).

HK ₁The peptase S that belongs to the serine stretch protein enzyme ₁Family's (or claiming the kallikrein subtribe).It can specificity hydrolysis lower molecular weight or high molecular weight kininogen (Kininogen) protein molecular in Met-Lys or the peptide bond between the Arg-Ser amino-acid residue, discharge kassinin kinin Lys-bradykinin or Kallidin respectively.These hydrolysis discharge kassinin kinin will be to blood pressure, electrolyte balance, inflammatory reaction and the cell proliferation performance regulating effect of body.It should be noted hK especially ₁Can preferentially cut among the small molecules chromogenic substrate S-2266 (Val-Leu-Arg-pNA)-Arg-pNA between peptide bond, this also is to adopt S-2266 to measure natural hK ₁And r-hK ₁Active basis.(Gurunathan?Laxmikanthan.etal?1.70?A ⁰?X-RayStructure?of?Human?Kallikrein?1：Structural?Changes?Upon?Peptide?Inhibitor/SubstrateBinding?Proteins.Structure，Function，and?Bioinformatics?58：802-814，2005)。

HK ₁Content in human body is the highest, after its metabolism major part again with complete activated glycoprotein form through renal excretion.At present, the hK of separation and purification from people's urine ₁Be used for clinical practice.For example, the general company in sky, Guangzhou to obtain the Human Urinary Kallidinogenase that SFDA produces certification (trade(brand)name ' Kai Likang  ', injection You Ruikelin) in April, 2005 be exactly the biological extraction product of urinating from the people.People's hK ₁Belong to thermostability strand glycoprotein, because glycosylated degree difference, so the also bigger (MW27～40kDa) of molecular weight variation range.

Both at home and abroad to adopting engineered method to produce hK ₁Work all carried out many researchs.The investigator of Germany just carried out that expressing human sialisterium hK (is hK in E.coli in 1989 ₁) research work (Angermann, A.etal Cloningand expression of human salivary-gland kallikrein in E.coli.Biochem J 1989,262 (3): 787-793), but the expression output in every liter of fermented liquid has only 200～500 μ g, and Jing Wang etc. are at the U.S. (Jing Wang, Julie Chao etal Purification and characterization of recombinant tissue kallikrein from E.coliand yeast.Biochem.J.1991 is 276:63-71) with E.coli and cereuisiae fermentum (S.cerevisiae) system expression type kallikrein (the similar hK of rat tissue ₁), and carry out separation and purification in conjunction with the Aprotinin-affinity media with DEAE-Sepharose CL-6B, the activity of rat kallikrein that found that the many methionine(Met) (Met) of N-end that E.coli expresses is with natural not difference.But when utilizing yeast α-signal peptide secreting, expressing rat kallikrein, some expression product can not excise signal peptide smoothly, so active kallikrein output extremely low (about 0.5～1mg/L fermented supernatant fluid).In addition, they are inclusion bodies the also mostly of E.coli expression, and it is very low to express output, adopts radio immunoassay to carry out quantitatively, and the output of inclusion body approximately has only 30～50mg/L fermented liquid.1993, (the GeorgFertig etal Biotech-nological aspects of the production of human pro-kallikrein using theAcNPV-baculovims-expression system Cytotechnology 11:67-75 such as investigator Georg Fertig of Germany, 1993) use insect karyomorphism polyhedrosis virus (AcNPV) expression system at expressed in insect cells people prekallikrein (Human Pro-kallikrein, Pro-hK ₁), after 13 days, the Pro-hK1 that the fermentation of 5L jar is obtained is converted to the also nearly 7960U of activity unit's ultimate production in cultured continuously in the high expressing cell strain that filters out.Afterwards, (Lu HS such as Lu, Hsu YR, etal Isolation andcharacterization of human tissue kallikrein produced in E.coli:biochemical comparison to theenzymatically inactive prokallikrein and methionyl kallikrein.Protein Expr Purif.1996,8 (2): 215-26.) expressed Pro-hK at E.coli ₁With Met-kallikrein (Met-hK ₁), Pro-hK ₁After cutting steps such as activation, chromatographic separation purifying through expression, inclusion body dissolving, protein folding renaturation, thermolysin enzyme substantially, obtained to have same biologic activity hK with natural product ₁Pro-hK ₁And Met-hK ₁Though structure and hK ₁Similar, but biologic activity all do not had, and as if this be conflicting with investigators' such as front Jing Wang result.Simultaneously, these investigator (LuHS, etal Purification and characterization of human tissue prokallikrein and kallikrein isoformsexpressed in Chinese hamster ovary cells.Protein Expr.Purif.1996,8 (2): 227-37.) genome total length prekallilkrein gene (Preprokallikrein, the Prepro-hK of expressing human in CHO again ₁), the proenzyme of this non-activity of reorganization prekallikrein is secreted in the fermented liquid in a large number, in purge process, and after process thermolysin enzyme is cut activation, just can be with Pro-hK ₁And hK ₁Purifying comes out respectively.The rhK that is obtained ₁With natural product relatively, its specific activity is obviously difference not, but Pro-hK ₁Or hK ₁Molecule itself and heterogeneity, these proteic molecular weight and electric charge have difference.The investigator infers it is due to the sialic acid residues number difference of the glycosylation modified degree of N-of molecule and sugar chain.

Relevant hK ₁The breakthrough work that genetically engineered is produced should be (Hedy Chan, etal Expressionand Characterization of Human Tissue Kallikrein Variants.Protein Expr ﹠amp such as Hedy Chan; Purif.1998 12:361-370) with three kinds of prekallikrein varients (Pro-hK1) gene secreting, expressing in methanol yeast (Pichia pastoris) of people, has obtained Pro-hK ₁Albumen through the trypsinase cutting, just can obtain hK again ₁The investigator adopts the PCR method to angle out Pro-hK from the cDNA library of human body different tissues ₁Gene has obtained the slightly different Pro-hK of three seed amino acid residues ₁Gene (variation is that the actual difference that just has is determined as yet in the point mutation that produces in the PCR process or the tissue in this).All wanting of research report before the rate ratio that methanol yeast is expressed is high many, reaches about 30mg/L fermented supernatant fluid, but the rhK that secreting, expressing goes out ₁Two bands are arranged, the product heterogeneity when the SDS-PAGE electrophoresis.

At home, Chinese Academy of Sciences's Dalian physical chemistry the investigator wait and to have described (Yang Q.etal Purification of humantissue prokallikrein excreted from insect cells by liquid chromatography.J Pharm Biomed Anal.2005,39:848-52.) with three the step liquid chromatographies, purifying is by the Pro-hK of insect cell expression ₁, yield is 57%, product purity is 95%.But this work also can only be regarded the extension of investigator's work such as Georg Fertig as.But, up to the present, with the product of being used for the treatment of property of the composition medicine of insect cell expression report not still, so the work of this respect does not still possess medicinal use at present.

As fully visible, the research that external these carry out in E.coli, CHO, insect cell, cereuisiae fermentum or methyl alcohol mother all is Pro-hK ₁Or Prepro-hK ₁Or Met-hK ₁Be expressed as the master, express hK and carry out direct secretion ₁During proteic work, find that signal peptide sequence can not excise smoothly.Except Hedy Chan etc. had obtained the high slightly engineering bacteria of output, other investigator's work all belonged to pure research character basically, from its output, preparation cost and aspect such as active, also all showed a large amount of preparation rhK ₁Very difficult.Hedy Chan etc. obtains Pro-hK earlier ₁, cut the processing back at enzyme and obtain hK ₁, although they find Pro-hK ₁Some can be directly with hK when secretion ₁The form secretion, but some is with Pro-hK ₁Form is secreted in the fermentation supernatant.Therefore, this phraseology does not possess productive value from output and purge process yet.

Though domestic investigator has also carried out expressing among the E.coli hK ₁Research, (purifying and the activation analysiss " the Chinese biological goods are learned magazine " 18 (1): 33～35 of human tissue kallikrein maturation proteins such as Li Tiyuan such as Li Tiyuan as Guangzhou, 2005), Du's a kind of jade, Study on expression of human pancreatic kininogenase " Chinese Pharmaceutical Journal " 38 (6): 465～467 such as Lee's body is far away, 2003) use the pMBP carrier, in E.coli, with hK ₁Same maltose binding protein (MBP) merges secreting, expressing, under the condition of 6L jar fermentation, has only obtained 24mg MBP-hK altogether ₁Fusion rotein through the zymoplasm cutting, could obtain active hK ₁These researchs are the same with external work in E.coli, not only express yielding poorly, and also do not solve issues of purification.Yuan Xin waits the (secreting, expressing " Beijing University of Chemical Technology journal " 31 (6) of the clear Chen Jing spring human pancreas kininogenase of Yuan Xin in pichia spp: 33～35,2004) directly express hK in methanol yeast expression expression system clearly ₁, hK in the fermentation supernatant ₁The about 40mg/L of output, and from the information that the investigator provides, do not know its expression product method for determination of amount yet, there is not the follow-up sex work of this research yet.Comprehensive at present relevant hK ₁Research, all r-hK ₁Preparation have the engineering strain of facing to express at the bottom of the output, product heterogeneity, purifying process complexity, the effectively very low shortcoming of yield.

This patent is according to the people hK that has announced among the Genebank ₁The gene order data, the 5`-and the 3`-primer of synthetic its mature protein gene sequence angle from commercial people's kidney cDNA and have got hK ₁Gene.This gene is inserted between the restriction enzyme XhoI and NotI site of pPICZ α A expression vector of Invitrogen company, behind the linearization process expression vector, electricity transforms methanol yeast host bacterium X33, screening positive clone on the YPD+Zeocin resistant panel, and the engineering strain that has obtained high expression level is screened in operation routinely.This bacterial strain can utilize the yeast α-signal peptide secreting, expressing r-hK of carrier self ₁Albumen, and do not find that the situation that α-signal peptide can't excise takes place, and expresses output up to 1.25～1.35g/L fermented supernatant fluid, r-hK ₁Though product has the difference of glycosylation modified degree, proteic N-, C-hold homogeneous, can obtain the r-hK of degree of glycosylation unanimity by purge process ₁Albumen.

Three, summary of the invention

1.hK ₁The acquisition of gene and pPICZ α-hK ₁The structure of expression vector

The sequence information of people's kallikrein-1 (the Homo sapines Kallikrein-1) gene (AY094609, NM-002257, BC005313, X13561, AY890098, AY890097, AY703451) that has obtained among the comprehensive GeneBank, design amplification hK ₁The 5`-of gene and 3`-primer are template with people's kidney cDNA library of Panomics company, have obtained people hK with the PCR method ₁Gene.

For goal gene correctly being inserted in the pPICZ α A carrier, 5 '-primer has XhoI site (CTCGAG), and at XhoI site and hK ₁The codon AAAAGA that adds the recognition sequence Lys-Arg correspondence of Kex2 proteolytic enzyme before the gene, the 3`-primer is to add terminator codon TAA and restriction enzyme NotI site (GCGGCCGC) after the codon of last amino-acid residue of carboxylic end of hK1, so, the hK that goes out of PCR ₁Gene just can be inserted between the XhoI-NotI site of pPICZ α A carrier through XhoI and the two backs of cutting of NotI, has so just obtained secreted expression carrier pPICZ α-hK1.

2. the screening of engineering strain

Correct pPICZ α-hK is confirmed as in order-checking ₁Expression vector SacI linearization process, electricity changes the hK after the linearizing ₁Expression vector is spread YPDZ (YPD+1.5%Agar+500 μ g/ml Zeocin) flat board, screening positive clone to methanol yeast X33 competent cell.With YPD nutrient solution propagation positive colony, about 6～8 hours, when thalline OD500 reaches 2～3, preserving a bacterium liquid is primordial seed, and all the other bacterium liquid 4000rpm are centrifugal, and supernatant is frozen to compare, and will change inducing culture liquid BMMY over to after the thalline suspension, the 300rpm shaking culture, 12 hours benefits of every mistake anhydrous methanol to the final concentration of methyl alcohol is 0.5%, so keeps the methanol induction state about 48 hours.Compare with supernatant before inducing, whether electrophoresis detection has target protein hK ₁Band occurs.Simultaneously, according to hK ₁Activity change is measured in the standard method of determination of activity (seeing embodiment 2), and according to active difference, screens the more positive colony of more number, so that obtain the r-hK of high expression level ₁Engineering strain.

3. zymotechnique determines

The fermentation of methanol yeast can be divided into the growth of growing microorganism, speed limit and these three complementary stages of abduction delivering, and the key parameter of this three phases controlled well just can obtain the high expression level of target protein.

The growing microorganism stage: the temperature of whole fermentation stage is 30.0 ℃.With ammoniacal liquor the pH value of initial total salt substratum is adjusted to 5.0, initial mixing speed 300rpm, air flow 0.5vvm, dissolved oxygen DO100% carries out growing microorganism and cultivates.After the glycerine in the initial substratum in the jar exhausted, the DO value can sharply rise (DO Spike), continued supplementary carbon source (glycerine or glucose etc.) and improved cell density, at this moment, promptly entered the speed limit growth phase of fermentation.

Speed limit growth phase: keep DO 〉=20%, continue the stream glycerol adding, make the thalline weight in wet base in the fermented liquid reach 180～200g/L.After the thalline weight in wet base reaches requirement, just use ammoniacal liquor, be adjusted to 6.0 on the pH value with fermentation, prepare to enter the abduction delivering stage.

The abduction delivering stage: suspend the carbon source supply, hungry thalline begins supply methyl alcohol after 30 minutes again, and slowly increase the amount of methyl alcohol, after treating that thalline adapts to, keep on the level basis of DO value 〉=20% and add inductor methyl alcohol, and keep this state to fermentation ends with maximum rate.

4. the activity determination method of people's kallikrein

4.1. the principle of measuring

HK ₁Energy specificity hydrolysis chromophoric substrate S-2266 (Val-Leu-Arg-pNA, Chromogenix) in the molecule-chemical bond between the Arg-pNA discharges pNA, the characteristic absorption peak of free pNA molecule is 405nm, the free pNA amount (A405 value) that discharges in unit time is directly proportional with the activity of enzyme, so, just can calculate hK according to the A405 value ₁Activity.

4.2. measure agents useful for same and solution

Carrying out the used reagent of this mensuration has: chromophoric substrate S-2266 solution (2mM), dilution buffer (20mMTris-Cl, pH8.0), reaction buffer (0.2M Tris-Cl, pH8.0) and reaction terminating liquid (50% acetum).

4.3. the process of determination of activity

According to the testing sample concentration of estimating, use 20mM Tris-Cl, the pH8.0 damping fluid is diluted to a series of multiples with testing sample, as 100,200,300 etc.In the 2ml of cleaning cillin bottle, add 400ul 0.2M pH8.0 Tris-Cl damping fluid, add the sample solution to be determined after 20 μ l dilute again, to then add 20 μ l dilution buffer in the same old way, behind the mixing, 37 ℃ of water bath heat preservations made in 5 minutes respectively measures solution temperature unanimity in the pipe, respectively adds 40 μ l chromophoric substrate S-2266 more respectively, mixing, accurately timing, and in 37 ℃ of water-baths 15 minutes.Then, add 40 μ l, 50% acetic acid solution termination reaction in each response sample, divide with contrast spectrophotometer is returned to zero, measure absorption value A405 value at 405nm wavelength place, the A405 value should then not increase or reduce extension rate and carry out again between 0.1～0.2 when this scope.

4.4. the calculating of measurement result

The A405 molar extinction coefficient of free type pNA is 9,600 L mol ^-1Cm ^-1, according to bK ₁The implication of activity unit: 1PNAU is meant at 37 ℃, Tris-Cl, and in the pH8.0 buffer system, the enzyme amount that can hydrolysis substrate S-2266 in 1 minute discharges the free pNA of 1 μ mol is a hK ₁Activity unit (PNAU).According to lambert-Bill (Beer-Lambert) law, hK in every milliliter of testing sample then ₁Activity unit can calculate by following formula:

The PNAU/ml=0.1736*A405* extension rate

Measure hK with the S-2266 chromophoric substrate ₁Activity is the basis of whole research work, and employing present method can be quickly and accurately to hK in the sample to be determined (fermented liquid, stoste, purifying middle sample etc.) ₁Activity is carried out accurately quantitatively.If r-hK ₁Accurate specific activity be 5.2PNAU/mg, in the time of will adopting this method to measure so, should be with r-hK to be determined ₁The protein concentration of sample is diluted in 3.2～6.5 μ g/ml and (is equivalent in 0.017～0.034PNAU/ml) scope, so just can reduces the workload of dilute sample, be convenient to obtain fast the result.

Description of drawings

Fig. 1. 6 high anti-Zeocin clones to primary dcreening operation on 24 orifice plates in the test tube carry out abduction delivering

Swimming lane 0. is the fermentation supernatant before the abduction delivering; The clone of swimming lane 1～6. high anti-Zeocin resistance (500 μ g/ml) induces 48 hours result.As seen, during the SDS-PAGE of reduction (R) and non-reduced (NR), the mobility of r-hK1 has difference, and two close bands are arranged.M：LMW?Marker?Kit(Amersham?Pharmacia?Biotech)：97.0kDa、66.0kDa、45.0kDa、30.0kDa、20.1kDa、14.4kDa。

Fig. 2 .30L fermentation inducement stage produces r-hK ₁SDS-PAGE reduction electrophoresis

The hours of the numeral methanol induction below the sampling in 8 hours of the every interval of whole induction period, each swimming lane.M：LMWMarker?Kit(Amersham?Pharmacia?Biotech)：97.0kDa、66.0kDa、45.0kDa、30.0kDa、20.1kDa、14.4kDa。

Fig. 3 .Phenyl-Sepharose FF chromatography (A) and Superdex75 sieve chromatography (B) purifying

A: use 20mM PB, 1.0M (NH ₄) ₂SO ₄, pH6.0 damping fluid balance Phenyl-Sepharose FF chromatography media.1. with 200mM PB, pH6.0 and 3M (NH ₄) ₂SO ₄Mother liquor adds to hK ₁In the fermented liquid, making it is 20mMPB at the fermented liquid final concentration, 1.0M (NH ₄) ₂SO ₄, pH6.0, use again 0.45 μ m membrane filtration sample solution; 2. effluent liquid; 3.20mM PB, 0.7M (NH ₄) ₂SO ₄, elution peak during pH6.0; 4.20mM PB, 0.35M (NH ₄) ₂SO ₄, elution peak during pH6.0, the wherein bigger than normal and low slightly hK of molecular weight ₁Respectively account for about 50%; 5.20mM PB, the pH6.0 elution peak.

The balance of B:Superdex75 sieve chromatography and elution buffer are 20mM PB, 0.15M NaCl, pH7.5, the elution peak that occurs during to the 0.5V column volume carries out Fractional Collections, is divided into 1., 2., 3., 4., 5. and 6. six sections, wherein 1. segment molecule amount maximum, amount is few, mass spectrum r-hK ₁The actual molecular weight that-B measures is 32871.16Dalton, and 4. section is hK ₁The main peak part, amount is maximum, mass spectrum r-hK ₁It is 28975.79Dalton that-A measures its actual molecular weight.M：LMW?Marker?Kit(AmershamPharmacia?Biotech)：97.0kDa、66.0kDa、45.0kDa、30.0kDa、20.1kDa、14.4kDa。

Fig. 4. gel permeation chromatography is purified into different glycosylation degree r-hK ₁Mass spectrum

R-hK ₁-A is electrophoresis molecular weight r-hK on the low side ₁, ratio is the highest in the albumen of whole secreting, expressing, r-hK ₁The higher part of molecular weight when-B is the SDS-PAGE electrophoresis, proportion is lower.M：LMW?Marker?Kit(AmershamPharmacia?Biotech)：97.0kDa、66.0kDa、45.0kDa、30.0kDa、20.1kDa、14.4kDa。

Fig. 5 .r-hK ₁The isoelectric point determination result

Each pI standard protein molecule of A and C among the swimming lane M～L representative is: A.Amylglucosidase (3.50); C.Trypsininhibitor (4.55); D.b-Lactoglobulin a (5.20); E.Bovine carbonic anhydrase b (5.85); F.Humancarbonic anhydrase b (6.55); G.Myoglobin, acidic band (6.85); H.Myoglobin, basic band (7.35); I.Lentil lectin, acidic (8.15); J.Lentil lectin, middle (8.45); K.Lentil lectin, basic (8.65); L.Trypsinogen (9.30).Swimming lane r-hK ₁-A is the low protein molecular of degree of glycosylation; Swimming lane r-hK ₁-B is the high protein molecular of degree of glycosylation.The mass spectrum of r-hK1-A and r-hK1-B correspondence such as Fig. 4.

Fig. 6 .PNGaseF removes r-hK ₁SDS-PAGE and the mass spectrum of N-after glycosylation modified

A:r-hK ₁Be when not carrying out the desugar processing, De-r-hK ₁Be the r-hK after PNGaseF handles ₁Molecular weight has obviously diminished as can be seen.B: De-r-hK1 is carried out mass spectroscopy, determine that its MW is 26404.35Dalton, very identical with the MW that calculates by the theoretical aminoacid sequence of r-hK1.

Four, embodiment

Embodiment 1

HK ₁The structure of engineering strain

1.hK ₁The acquisition of gene and expression vector pPICZa-hK ₁Make up

With reference to the relevant hK that announces among the GeneBank ₁Gene complete sequence data, ripe hK can increase to design and synthesize (worker is given birth in Shanghai) ₁Two primer Kal5 of gene (the 5`-primer, SEQ-3) Kal3 (the 3`-primer, SEQ-4):

Kal5(AA07624)：

5`-cat ctc?gag?aaa?aga?att?gtg?gga?ggc?tgg?gag?tgt?gag-3`

Kal3(AA07625)：

5`-cat? gcg?gcc?gct?tag?gag?ttc?tcc?gct?atg?gtg?tcc?tc-3`

(P/N:7202 L/N:P0260602) is template, just obtains hK through PCR with people's kidney cDNA library of Panomics company ₁Gene.Primer Kal5 will be at hK ₁Gene 5`-end adds the codon AAA AGA of the recognition sequence Lys-Arg correspondence of DNA restriction enzyme XhoI site (CTCGAG) and Kex2 proteolytic enzyme, and this will guarantee the hK of insertion <sub>1</sub>Can excise α-signal peptide smoothly during the gene secreting, expressing, obtain to have the hK of natural N-terminal sequence <sub>1</sub>Protein molecular; Primer Kal3 adds terminator codon TAA and DNA restriction enzyme NotI site (GCGGCCGC) at the 3`-end.

(Pyrobest) carries out pcr amplification with archaeal dna polymerase, and system is as follows:

Human?kidney?cDNA------------------1μl

Kal5 (5`-primer)---------------------2 μ l

Kal3 (3`-primer)---------------------2 μ l

dNTP?Mix?-------------------------1μl

10x?Pyrobest?Buffer----------------------2μl

Pyrobest------------------------------0.25μl

ddH ₂O?--------------------------------1175μl

Total 20 μ l

The PCR condition: 94 ℃ of sex change 3 minutes, the mode by 94 ℃, 30 seconds/55 ℃, 30 seconds/72 ℃, 1 minute kind circulates 30 times again.PCR gets 2 μ l amplified productions in 1.5% agarose electrophoresis after finishing, and can obtain length and just show the PCR success for being about the 750bp fragment.

The PCR product is reclaimed test kit (Sangon) purifying with post,, reclaim hK through glue with restriction enzyme XhoI and the two PCR products that reclaim through post of cutting of NotI ₁Gene.PPICZ α A carrier (Invitrogen) is cut with XhoI and NotI are two, and the big fragment that reclaims carrier is as the carrier that inserts goal gene, with the hK that reclaims ₁Gene fragment is with setting up following ligation with pPICZ α carrier:

PPICZ α carrier (XhoI-NotI)-----------------2 μ l

HK ₁Gene (XhoI-NotI)------------------4 μ l

10×Ligation?Buffer------------------------1μl

T ₄DNA Ligase (ligase enzyme)-------------0.5 μ l

ddH ₂O---------------------------2.5

Total 10 μ l

Ligation was carried out about 1 hour under 22 ℃ of conditions, got above-mentioned connection product 5 μ l and CaCl ₂The careful mixing of TOP10F ' competence 100 μ l of method preparation, 4 ℃, 30 minutes, 42 ℃, 90 seconds kinds of heat-shocked, put on ice 3～5 minutes, add 500 μ lLB nutrient solutions then,, get 100 μ l bacterium liquid and coat on the flat board that contains LZ (LB+Zeocin25 μ g/ml)+1.5% agarose in 37 ℃ 200 rev/mins cultivations 45 minutes, 37 ℃ of incubated overnight transform successful positive colony with growing.Therefrom at random 6 single colony inoculations of picking in 5ml LZ nutrient solution, 37 ℃ of shaking table overnight incubation, with plasmid DNA extraction agent box (BioAsia company) extracting plasmid, cut screening with XhoI and NotI are two, get and can enzyme cut out the segmental cloning and sequencing of about 750bp and prove conclusively sequence.Sequencing result such as SEQ-1, its corresponding amino acid sequence such as SEQ-2.This is as broad as long with the sequence of having announced among the GenBank.

2. engineering strain obtains

Prepare the X-33 competent cell according to method among the P.methanolica Expression Kit of Invitrogen company, plasmid expression vector is cut with the SacI enzyme and is carried out linearizing, phenol-chloroform extrct Deproteinization, linearized vector and the X33 competent cell mixing of ethanol sedimentation after with deionized water dissolving, electricity transforms, coat YPD (1% yeast extract that contains Zeocin 500ug/ml, 2% polyprotein peptone, 2% glucose) on the flat board, cultivated 2～3 days for 30 ℃, up to there being yeast list colony growth to occur.According to the description among the Easyselect Pichia Expression Kit, at first screen recon by improving the antibiotic resistance of Zeocin, 100 recons of picking are inoculated in 24 orifice plates of 400 μ l YPD (containing Zeocin 600 μ g/ml) nutrient solution, 30 ℃ of shaking table overnight incubation, with the high anti-Zeocin hole of sifting out reserve seed for planting (6 satisfactory clones) filter out altogether and be transferred to and add 2ml YPD nutrient solution in the test tube and continue propagation, centrifugal collection thalline after 24 hours, with 30 ℃ of abduction deliverings of the BMMY substratum that contains methyl alcohol 0.5%, it is 0.5% that 24 hours interpolation methyl alcohol makes its final concentration in bacterium liquid, kept this induction state 48 hours, centrifugal collection bacterium liquid supernatant, contrast before and after the SDS-PAGE electrophoresis detection is induced has the 30kDa protein band to express in the fermentation supernatant after finding to induce.Be difficult to accurately judge expression output because the electrophoresis observation strip type of albumen changes,, make blank, accurately measure with chromophoric substrate S-2266 and induce r-hK in the secondary fermentation supernatant with supernatant before inducing so also adopt the method for describing in the front summary of the invention 4. simultaneously ₁Activity unit's output.Determine the wherein hK of plant height expression at last ₁Engineering strain (Fig. 1) conduct fermentation usefulness, and set up three grades of seed banks.

Embodiment 2

R-hK ₁The fermentation of engineering bacteria

1. seed liquor is prepared

Get the frozen pipe of work seed glycerine, get 1ml after the thawing and be seeded in the 500ml YPD substratum (1% yeast powder, 2% peptone, 2% glucose),, cultivate 30 hours in the shaking table of 300rpm to OD at 30 ℃ ₆₀₀Value is 6.0 ± 1.0, and microscopy normally just obtains jar inoculation usefulness on the seed liquor.Preparation is fermented with basic salt culture medium BSM ₁(K ₂SO ₄60.7 gram, MgSO ₄24.2 gram, CaSO ₄2H ₂O 3.9 grams, H ₃PO ₄89 milliliters, KOH 13.8 gram, 14 milliliters of PTM1, glycerine 400 grams, 2 milliliters of bubble enemies are example with 10L, are example to prepare 1 liter micro-substratum PTM1, wherein the content of each component is: CuSO ₄5H ₂O 6.0 grams, NaI 0.008 gram, MnSO ₄3.0 gram, NaMoO ₄0.2 gram, H ₃BO ₃0.02 gram, ZnSO ₄20.0 gram, CoCl ₂0.5 gram, FeSO ₄7H ₂O 65.0 grams, vitamin H 0.2 gram, H ₂SO ₄5 milliliters, adding the water constant volume is 1 liter) after carry out reality jar sterilization.Sterilising conditions is 121 ℃, 20 minutes, is chilled to 30 ℃ after disappearing.By 1: 15 (V/V, seed liquor: basis jar basic nutrient solution BSM ₁) the ratio inoculation.

2. fermenting process

The leavening temperature in growing microorganism stage at initial stage is 30.0 ± 0.5 ℃, pH5.00 ± 0.05, and initial rotating speed 300rpm cultivates, air flow 0.5vvm, DO value 100% is added PTM ₁About about 20 hours of this stage, keep the DO value and be not less than 30%, when carbon source is exhausted, oxygen dissolving value promptly rises, the thalline weight in wet base reaches about 100g/L, enter the speed limit growth phase this moment, and the beginning in this stage is during 2 hours, and adding concentration with 240ml/ hour speed is 50% glycerine solution.After the feed supplement 2 hours, be adjusted to 360ml/ hour on the feed rate.By regulate mixing speed, air flow quantity, tank pressure (＜0.8bar) the DO value is maintained be not less than 20% level.Add about 4 hours, during the about 160～180g/L of thalline weight in wet base, stop feed supplement, the DO value rises.The DO value rises and to show that carbon source exhausts in the substratum, and the inductor methyl alcohol of should carbon source holding concurrently carries out feed supplement, and this just enters the key abduction delivering stage in stage of fermentation: with ammoniacal liquor the pH value is controlled at 6.20 ± 0.05, begins to add methyl alcohol.The initial add-on of methyl alcohol was controlled at 30ml/ hour, slowly increased the add-on of methyl alcohol, was 60ml/ hour with the feed supplement speed setting after about 2 hours.Inducing after 4 hours the methanol feeding speed setting is 120ml/ hour, methanol induction success this moment, and thalline can normally utilize methyl alcohol as carbon source at a high speed.Keep the DO value and be not less than 20%, 30 ℃ of temperature, pH value are 6.20 ± 0.05, carry out fermentation expression, between whole yeast phase, get sample one time in per 8 hours, measure active (table 1) and SDS-PAGE electrophoresis (Fig. 2).Finish fermenting process when inducing 60 hours, centrifugal collection supernatant, SDS-PAGE electrophoresis and S-2266 chromophoric substrate method detect expresses output.

Whole fermentation inducement is a bioassay standard with 1 milliliter of fermented liquid in the stage, measures and enters methanol induction during the stage, and thalline weight in wet base increase in the fermented liquid, fermentation supernatant reduce expression product r-hK ₁Proteic remarkable increase situation.As seen, be not induction time the longer the better because sampling can reduce the fermented liquid cumulative volume in the fermenting process, thalline rolls up can make supernatant significantly reduce and induction time can make foreign protein increase considerably r-hK for a long time ₁Degraded increases, and the induction time of weighing after these factors fermentation is decided to be 50～60 hours.

Table 1. fermentation inducement stage thalline weight in wet base, supernatant volume and expression rate ratio are

The methanol induction time (hour)	1ml fermented liquid (samplings of different induction times)
					Supernatant volume (μ l)	Thalline weight in wet base (μ g)	Active (PNAU/ml)	Gross activity (PNAU)
	0	875	168	0.04					0.035
8					838	184	0.89	0.746
	16	806	216	1.42					1.145
24					781	276	2.0	1.562
	32	744	335	3.0					2.232
40					720	345	3.8	2.736
	48	670	367	4.8					3.216
56					650	399	6.2	4.030
	64	620	420	6.7					4.154

Embodiment 3

R-hK ₁Proteic purifying

Purge process was made up of hydrophobic, anionresin and three steps of gel permeation chromatography, and wherein hydrophobic chromatography is selected Phenyl-Sepharose FF, and anionic exchange medium is Q-Sepharose FF, gel permeation chromatography employing Superdex75.

Detailed process is as follows:

1. the pre-treatment of fermented liquid

In fermented supernatant fluid, add 0.2M PB, pH6.0 and 3.0M (NH ₄) ₂SO ₄Solution to its final concentration in fermented liquid is respectively 20mM and 1.0M, regulates pH value to 6.0, leave standstill after 1 hour 0.45 micron membrane filtration contain 20mMPB, 1.0M (H ₄) ₂SO ₄, the processing secondary fermentation liquid (sample solution) of pH6.0 just can carry out the chromatography process.

2.Phenyl-Sepharose FF hydrophobic chromatography

With Phenyl Sepharose FF chromatography column on the fermented liquid after the above-mentioned processing, column type is Index70/500, and column volume is 500ml, and level pad is 20mM PB, 1.0M (NH ₄) ₂SO ₄, pH6.0, elution buffer are 20mM PB, 0.7M (NH ₄) ₂SO ₄, pH6.0,20mM PB, 0.4M (NH ₄) ₂SO ₄, pH6.0 and 20mM PB, pH6.0 collects each elution peak.Target protein r-hK wherein ₁Mainly at 20mM PB, in the pH6.0 elution peak (Fig. 3 A).

3.Q-Sepharose FF anion-exchange chromatography

Hydrophobic chromatography wash-out main peak is collected liquid use 1.0M NaOH to adjusted pH to 7.5, the dilution electricity is led≤8.0mS/cm last Q-Sepharose FF anion-exchange chromatography post, the post specification is 5 * 10cm, column volume is 200ml, level pad 20mM PB, pH7.5, elution buffer is 20mM PB, 0.2M NaCl, pH7.5 and 20mM PB, 0.5M NaCl, pH7.5 collects each elution peak.Target protein r-hK ₁Mainly at 20mM PB, 0.5M NaCl is in the elution peak of pH7.5.

4.Superdex 75 gel permeation chromatographies

That balance and wash-out adopt is 20mM PB, 0.15M NaCl, the pH7.5 damping fluid is pulled on Superdex 75 with anion-exchange chromatography elutriant branch, and the post specification is 6.0 * 60cm prepacked column, column volume is 1700ml, each applied sample amount is 5% (the about 85ml) that is no more than column volume, and Fractional Collections main peak, SDS-PAGE electrophoresis are determined to merge satisfactory part after the purity, filtration sterilization is r-hK ₁Stoste (Fig. 3 B).

Embodiment 4

R-hK ₁The character conclusive evidence

1.r-hK ₁Specific activity is measured

To the r-hK that is purified into ₁Carry out purity of protein (SDS-PAGE electrophoresis, RP-HPLC) and measure, purity is not less than 98% r-hK ₁Accurately measure concentration with the Lorry method, according to hK in the summary of the invention 4. ₁Activity determination method in describe, measure activity unit (PNAU) number with the S-2266 chromophoric substrate.With activity unit (PNAU)/protein concentration (mg), just can draw r-hK ₁Specific activity, actual result is about 5.2PNAU/mg.

2.r-hK ₁Proteic mass spectrum and N-end and C-terminal sequence are measured

The r-hK that the gel permeation chromatography Fractional Collections is purified into ₁Albumen is got on the low side higher as two groups with molecular weight of molecular weight, entrust Shanghai Inst. of Life Science, CAS biochemistry to carry out the n-end of albumen sequential analysis according to standard method with cell research institute proteomics research center, the result shows: though both have difference on molecular weight, but do not find significant difference when measuring its external activity with S-2266 chromophoric substrate method, the N-terminal sequence of their polypeptide chains is in full accord, and N-holds preceding 15 amino-acid residues to be: I-V-G-G-W-E-C-E-Q-H-S-Q-P-W-Q-.Utilize the carboxypeptidase hydrolysis method to measure this two groups of r-hK ₁Three amino-acid residues of carboxylic end be-E-N-S.This shows: we utilize the secreting, expressing r-hK of methanol yeast system ₁The time the described N-end of investigator heterogeneity phenomenon before not, the molecule heterogeneity of electrophoresis showed is due to the glycosylation modified degree difference, but can separate the different r-hK of degree of glycosylation by purifying ₁Protein molecular.

3.r-hK ₁Proteic isoelectric point determination

Adopt isoelectric focusing method to measure r-hK ₁Proteic iso-electric point, used pI standard protein are Broad pI Kit pH3～10 of GE Healthcare LifeScience company, high-glycosylation and low glycosylation modified r-hK ₁Proteic pI is not obviously difference also, and all in the region intermediate corresponding position, 3.5～4.5 position (Fig. 5) of pI standard protein, this shows the r-hK that methanol yeast is expressed ₁PI be about 4.0.

4.r-hK ₁Proteic de-glycosylation test

Infer hK by theoretical ₁Three possible glycosylation modified sites of N-type are arranged in the proteic aminoacid sequence, and whether these sites are at yeast secreted expression r-hK ₁All taken place glycosylation modified during albumen? according to hK ₁Proteic aminoacid sequence calculates the hK that does not modify ₁The molecular weight of polypeptide chain should be 26405.74Dalton, and the actual r-hK that is purified into ₁Proteic molecular weight and heterogeneity from SDS-PAGE electrophoresis (Fig. 1, Fig. 2, Fig. 3) as can be seen, but are r-hK with the mass spectroscopy purifying more accurately ₁Proteic molecular weight, according to Fig. 4 from 28975.79Dalton to 31871.16Dalton, the r-hK that expresses of methanol yeast secretion so ₁2570.05～5465.42Dalton of albumen net increase on molecular weight just should be the molecular weight that methanol yeast secretion adds up ornamental equivalent when albumen being carried out posttranslational modification when expressing, and that is to say that modifying the N-sugar chain that increases partly accounts for 8.86～17.15% of protein molecular.

Handle r-hK with the standard operation that PNGase F proteolytic enzyme (New England BioLabs) is pressed in the service manual ₁, the SDS-PAGE electrophoresis is removed r-hK behind the N-sugar chain as can be seen ₁Molecule has diminished (Fig. 6 A), will remove the r-hK of N-type sugar chain ₁(De-r-hK ₁) carrying out mass spectroscopy (Fig. 6 B), the molecular weight of this moment is 26404.35Dalton, very approaching with the 26405.74Dalton that theory calculates, this shows the r-hK that methanol yeast gives expression to ₁There is no other modification mode in the albumen, have only zymic N-type glycosylation modified.

Embodiment 5

R-hK ₁Influence to focal cerebral ischemia in rats

This test entrusts Second Military Medical University, PLA pharmacology religion research department to carry out, and PRELIMINARY RESULTS is as follows:

1. experiment material

1.1 medicine

Be subjected to reagent: lot number is 20060218 r-hK ₁Stoste, 98% of purity, protein concentration 4.8mg/ml, active concentration 25PNAU/ml.Face with preceding and be diluted to desired concn with physiological saline.Positive control drug: nimotop vial (Shandong XinHua Pharmacy stock Co., Ltd, lot identification mark 0411022) contains nimodipine 10mg in every 50ml injection liquid.Solvent control: 0.9% sodium chloride injection (Shanghai Baxter Healthcare Ltd., lot identification mark A6B11307).

1.2 reagent

TCC (TTC) (worker's biotechnology company limited, lot number 2803B19 are given birth in Shanghai) faces with preceding that to be made into concentration with the PBS damping fluid be 2%, and lucifuge is standby.

1.3 laboratory animal

Male SD rat, body weight restrains to 350 grams, available from Shanghai Slac Experimental Animal Co., Ltd. from 280.

1.4 instrument

Image analysis system (Germany, Leica Microsystems, Type DM LB2).

2. experimental technique

2.1 local cerebral ischemia model preparation

Get male rat, abdominal injection 15% Chloral Hydrate (300mg/kg) anesthesia.Dorsal position is fixed, neck median line otch, and along nutator inner edge separating muscle and manadesma, careful separation left carotid (CCA), external carotid artery (ECA) and internal carotid artery (ICA) and occipital artery, standby at CCA distal end and proximal part and ECA place hanging wire.Close ICA, proximal part ligation CCA, ECA then with the temporary transient folder of arteriole folder.Cut an osculum at distance CCA furcation 4mm place, will fasten line and be inserted into ICA, at this moment use to fasten gently and fasten line around the fine rule of CCA distal end.Touch with the ophthalmology tweezer and to fasten line, begin to calculate distance,, tightly fasten the fine rule of CCA distal end, the layer-by-layer suture otch when depth of penetration during at 20mm from vascular bifurcation.

2.2 jugular vein intubate administration

Separate external jugular vein, the threading twice are standby.The distal end ligation, proximal part bulldog clamp folder closes.Diagonal is cut a kerf, and the venous duct miter angle that will be full of soup or physiological saline inserts.Proximal part ligation conduit is removed bulldog clamp.Whether the pumpback inspection is successful slightly, causes back 30min by the administration of 0.1ml/100g body weight in the cerebral infarction model.Other wears a line in the conduit below, with the ligation of catheter tip vein, extracts conduit, layer-by-layer suture.Steam again and raise.Room temperature is strict controlled in 24～25 ℃.The decerebrate that breaks end behind the 24h is measured infarcted region area and infarcted region weight.

2.3 experiment grouping and dosage

Experiment divides three groups (model group, positive controls are subjected to the reagent group), every group of about 15 male SD rats.The model group modeling also gives isopyknic physiological saline 0.1ml/100g.Positive drug control group gives nimotop vial 0.5mg/kg.Be subjected to the reagent group to give r-hK ₁30 * 10 ^-3PNAU/kg, 0.1ml/100g.

2.4 observation index

Postoperative 24h observes and writes down the death condition of animal earlier, then the animal broken end is got brain, puts into not real estate group cerebral infarction per-cent n of dead count table 3. Buddhist nuns of-20 ℃ of ice tables 2.24 hour each treated animal peeling off complete brain.

The dead counting of each treated animal in the table 2.24 hour

Group	Total routine number	Dead routine number in 24 hours	Mortality ratio (%)
				Model group	14	4	28.6
The nimodipine group	5	0	0
				r-hK 1Group	11	1	9.1

Table 3. nimodipine group cerebral infarction per-cent

The rat numbering	Planimetry (%)	Weighting method (%)
			4	35.6	28.2
5	41.9	40.9
			9	11.7	12.3
23	17.1	8.50
			35	8.40	4.26

As shown in table 2, nimodipine group and r-hK ₁The rats death rate significantly is lower than model group in organizing 24 hours, prompting r-hK ₁All can reduce the death of acute cerebral infarction animal with nimodipine.

Table 4. model group cerebral infarction per-cent

The rat numbering	Planimetry (%)	Weighting method (%)
			11	52.3	58.6
13	59.0	55.4
			22	32.3	23.8
24	51.2	51.3
			25	46.9	45.3
27	13.6	12.3
			31	28.9	27.4
36	53.2	54.0
			39	50.9	53.1
41	33.9	30.3

Table 5.r-hK ₁Group cerebral infarction per-cent

The rat numbering	Planimetry (%)	Weighting method (%)
			1	40.5	41.7
6	24.7	23.0
			16	33.9	34.8
17	6.60	5.11
			28	18.5	22.7
29	4.60	3.40
			32	19.9	24.7
38	14.1	7.13
			42	8.70	10.6
46	5.10	5.55

Compare with model group: ^*P＜0.05, ^*P＜0.01.

Shown in table 6 is summed up, compare with model group, the positive control drug nimodipine can dwindle cerebral infarction dead band area and cerebral infarction dead band weight (P＜0.05) significantly.Be subjected to reagent r-hK ₁Compare with model group, can dwindle cerebral infarction dead band area and cerebral infarction dead band weight (P＜0.01) very significantly.The experimental result prompting: similar with nimodipine, r-hK ₁Acute cerebral ischemic infarction had therapeutic action.

DNA and proteinic sequence description

The data of SEQ-1 sequence

(1). sequence signature:

A. length: 714bp

B. type: Nucleotide

C. topology: linearity

(2). molecule type: people's tire kidney cDNA

(3). sequence description: SEQ-1

1?ATTGTGGGAG?GCTGGGAGTG?TGAGCAGCAT?TCCCAGCCCT?GGCAGGCGGC

51?TCTGTACCAT?TTCAGCACTT?TCCAGTGTGG?GGGCATCCTG?GTGCACCGCC

101?AGTGGGTGCT?CACAGCTGCT?CATTGCATCA?GCGACAATTA?CCAGCTCTGG

151?CTGGGTCGCC?ACAACTTGTT?TGACGACGAA?AACACAGCCC?AGTTTGTTCA

201?TGTCAGTGAG?AGCTTCCCAC?ACCCTGGCTT?CAACATGAGC?CTCCTGGAGA

251?ACCACACCCG?CCAAGCAGAC?GAGGACTACA?GCCACGACCT?CATGCTGCTC

301?CGCCTGACAG?AGCCTGCTGA?TACCATCACA?GACGCTGTGA?AGGTCGTGGA

351?GTTGCCCACC?CAGGAACCCG?AAGTGGGGAG?CACCTGTTTG?GCTTCCGGCT

401?GGGGCAGCAT?CGAACCAGAG?AATTTCTCAT?TTCCAGATGA?TCTCCAGTGT

451?GTGGACCTCA?AAATCCTGCC?TAATGATGAG?TGCAAAAAAG?CCCACGTCCA

501?GAAGGTGACA?GACTTCATGC?TGTGTGTCGG?ACACCTGGAA?GGTGGCAAAG

551?ACACCTGTGT?GGGTGATTCA?GGGGGCCCGC?TGATGTGTGA?TGGTGTGCTC

601?CAAGGTGTCA?CATCATGGGG?CTACGTCCCT?TGTGGCACCC?CCAATAAGCC

651?TTCTGTCGCC?GTCAGAGTGC?TGTCTTATGT?GAAGTGGATC?GAGGACACCA

701?TAGCGGAGAA?CTCC

The data of SEQ-2 sequence:

(1). sequence signature:

A. length: 238 amino acid

B. type: amino acid

C. topology: the unknown

(2). molecule type: protein

(3). sequence description: SEQ-2

1?IVGGWECEQH?SQPWQAALYH?FSTFQCGGIL?VHRQWVLTAA?HCISDNYQLW

51?LGRHNLFDDE?NTAQFVHVSE?SFPHPGFNMS?LLENHTRQAD?EDYSHDLMLL

101?RLTEPADTIT?DAVKVVELPT?QEPEVGSTCL?ASGWGSIEPE?NFSFPDDLQC

151?VDLKILPNDE?CKKAHVQKVT?DFMLCVGHLE?GGKDTCVGDS?GGPLMCDGVL

201?QGVTSWGYVP?CGTPNKPSVA?VRVLSYVKWI?EDTIAENS

The data of SEQ-3 sequence:

(1). sequence signature:

A. length: 39nt

B. type: Nucleotide

C. topology: linearity

(2). molecule type: artificial synthesized sequence

(3). sequence description: SEQ-3

1?CATCTCGAGA?AAAGAATTGT?GGGAGGCTGG?GAGTGTGAG 39

The data of SEQ-4 sequence:

(1). sequence signature:

A. length: 38nt

B. type: Nucleotide

C. topology: linearity

(2). molecule type: artificial synthesized sequence

(3). sequence description: SEQ-4

1?CATGCGGCCG?CTTAGGAGTT?CTCCGCTATG?GTGTCCTC 38

Claims (7)

1. one kind by methanol yeast (Pichiapastoris) system high efficiency secreting, expressing, and by fermentation, purge process preparation and contain glycosylation modified recombinant human kallikrein-1 (r-hK ₁).

2. according to the efficient secretory expression in the claim 1, r-hK in the fermentation supernatant that is obtained when being meant jar fermentation ₁Protein yield can reach 1.2g/L.

3. according to the prepared r-hK of claim 1 ₁Molecular weight ranges be 28～32kDa, whole molecule is a polypeptide chain that contains 238 amino-acid residues, it is I-V-G-G-W-E-C-E-Q-H-S-Q-P-W-Q-that its N-holds preceding 15 amino-acid residues, terminal 3 amino-acid residues of C-are-E-N-S, intramolecularly all is that the N-type sugar chain of methanol yeast is modified, wherein carbohydrate accounts for 8.86～17.16% of whole glycoprotein, and proteic iso-electric point (pI) is about 4.0.R-hK ₁Gene and aminoacid sequence be respectively SEQ-1 and SEQ-2.

4. according to r-hK prepared in the claim 1 ₁, the specific activity that adopts S-2266 chromophoric substrate method to determine is 5.2 PNAU/mg.

5. the fermentation according to indication in the claim 1 is to adopt the total salt substratum, and methanol induction is expressed r-hK ₁The time pH6.2, temperature is 30 ℃, induction time 50 hours.

According to the purifying of indication in the claim 1 be by hydrophobic chromatography (Phenyl-SepharoseFF) catch, anion-exchange chromatography (Q-SepharoseFF) removes foreigh protein removing and concentrate target protein r-hK ₁, and gel permeation chromatography (Superdex-75) Fractional Collections obtains high purity r-hK ₁This three steps chromatography purification process is formed.Wherein the suitableeest upward batten spare of Phenyl-Sepharose FF hydrophobic chromatography is 20mM PB, 1.0M (NH ₄) ₂SO ₄, pH6.0, the suitableeest elution requirement are 20mM PB, pH6.0, and the last sample specific conductivity of Q-Sepharose FF anion-exchange chromatography is not higher than 8.0mS/cm.

7. according to r-hK prepared in the claim 1 ₁Can significantly reduce the mortality ratio of rat cerebral infarction animal model.

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