CN1137261C - Punctiform aerogenic monad prolyl endopeptidase and its preparation method and application - Google Patents

Abstract

The present invention discloses new prolyl endopeptidase [EC3.4.21.26] which is the prolyl endopeptidse of aeromonas punctats subsp. Punctata. More specifically, the present invention discloses the amino acid sequence and the DNA coding sequence of the prolyl endopeptidase, an expression vector and an engineering bacterium for expressing the prolyl endopeptidase, a preparation method and the purpose of the prolyl endopeptidase.

Description

Punctiform aerogenic monad prolyl endopeptidase and method for making thereof and purposes

The present invention relates to genetically engineered and zymetology field.More specifically, the present invention relates to a kind of new prolyl endopeptidase [EC 3.4.21.26], the aminoacid sequence of this prolyl endopeptidase and dna encoding sequence are used to express the expression vector and the engineering bacteria of this prolyl endopeptidase and the preparation method of this prolyl endopeptidase and purposes.

Prolyl endopeptidase (Prolyl endopeptidase is called for short PEP) [EC 3.4.21.26] is serine protease (the Wilk S.LifeScience of proline(Pro) carboxy-terminal peptide bond in a kind of energy specificity hydrolysis small molecular weight polypeptide, 1983,33 (22): 2149).

As everyone knows, in 20 kinds of a-amino acids forming natural protein or polypeptide, proline(Pro) (Pro) is unique amino acid with imido structure, space conformation when its ring texture has influenced with other amino acid formation peptide bonds makes peptide bond or its other contiguous peptide bonds of containing Pro in the protein insensitive to extensive narrow spectrum proteolytic enzyme simultaneously.Nature has produced some during evolution specially at the proteolytic enzyme of Pro residue in the polypeptide, and these proteolytic enzyme even all there is very strong specificity the position of Pro in polypeptide with the configuration that becomes key, this fermentoid is called proline(Pro) specificity peptase, just prolyl endopeptidase (PEP).Prolyl endopeptidase can be used as a kind of molecular biological toolenzyme, and the enzyme that is used for protein sequencing, peptide spectrum analysis, specific site is cut, the modification of peptide section and processing etc.

Studies show that PEP is distributed widely in mammiferous various tissue, also be present in a few fungi, bacterium even the archeobacteria simultaneously, but its content be very low that the separation and purification difficulty has limited the research to its physiological function and character.Therefore abroad from meningitis septicopyemia Flavobacterium (Chevallier S, Goeltz P, Thibault P etal.J Biochem, 1992,267 (12): 8192), Aeromonas hydrophila (Kanatani A, Yoshimoto T, Kitazona Aet al.J Biochem, 1993,113:790), heat-resisting archeobacteria (Robinson K A, Bartley D A, Robb F T etal.Gene, 1995,152:123), human lymphocyte (Shirasawa Y, Osawa T, Hirashima A.JBiochem, 1994,115:724) with pig brain cDNA storehouse (Rennex D, Hemmings B A, Hofsteenge J etal.Biochemistry, 1991, cloned the PEP gene in 30:2195), carried out genetically engineered research.

Yet, also do not have the report of relevant Punctiform aerogenic monad prolyl endopeptidase up to now.

The purpose of this invention is to provide a kind of new prolyl endopeptidase, it is the prolyl endopeptidase of point-like aerogenesis Zymomonas mobilis.

Another object of the present invention provides a kind of dna sequence dna of this novel prolyl endopeptidase of encoding.

A further object of the present invention provides the method and the purposes of producing this novel prolyl endopeptidase.

In a first aspect of the present invention, a kind of isolating prolyl endopeptidase is provided, this enzyme is the prolyl endopeptidase of point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata).Preferably, this enzyme comprises the aminoacid sequence shown in the SEQ ID No.2.

In a second aspect of the present invention, a kind of separated DNA sequence is provided, this dna sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID No.2.Preferably, this dna sequence dna comprises the nucleotide sequence shown in the 1-2073 position among the SEQ ID No.1.

In a third aspect of the present invention, a kind of expression vector is provided, it contains the dna sequence dna that above-mentioned code book is invented novel prolyl endopeptidase.In addition, also provide described expression vector transformed host cells.Preferably, this host cell is a prokaryotic cell prokaryocyte, more preferably is intestinal bacteria.

In a fourth aspect of the present invention, a kind of method that produces prolyl endopeptidase is provided, this method comprises:

(a) will the encode nucleotide sequence of prolyl endopeptidase of shape point aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata) operationally is connected in expression regulation sequence, form the prolyl endopeptidase expression vector of some aerogenesis Zymomonas mobilis point-like subspecies, described nucleotide sequence coded polypeptide with the aminoacid sequence shown in the SEQ ID No.2;

(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of prolyl endopeptidase;

(c) be fit to express under the condition of this prolyl endopeptidase polypeptide the reconstitution cell in the culturing step (b);

(d) isolate and have an active polypeptide of prolyl endopeptidase of aerogenesis Zymomonas mobilis point-like subspecies.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 3.4kb, and wherein the detailed sequence of open reading frame is seen SEQ ID NO.1, and promptly open reading frame is positioned at 1-2073 position Nucleotide.

Fig. 1, the pAPEP01 construction of recombinant plasmid.

Fig. 2, the pAPEP11 construction of recombinant plasmid.

Fig. 3, the restriction map of 3.5kb HincII/EcoRI active fragments.

Fig. 4, the nucleotide sequence of PEP gene.

Fig. 5, the aminoacid sequence of PEP genes encoding.

Fig. 6, the PEP aminoacid sequence in six kinds of sources compares, and * represents very conservative amino acid, and expression is the amino acid of expression relatively.APEP, the PEP in aerogenesis Zymomonas mobilis point-like subspecies source; AHPEP bites aqueous vapor pseudomonas PEP; FPEP, meningitis septicopyemia Flavobacterium PEP; PPEP, pig brain PEP; HPEP, human lymphocyte PEP; PFPEP, furious fireball bacterium PEP.

Fig. 7, the structure of recombinant plasmid pGEM-PEP.

Fig. 8, the electrophorogram of HPLC gel filteration determining PEP molecular weight.

Fig. 9, the mensuration of PEP iso-electric point.

Figure 10, pH is to the graphic representation of PEP activity influence.

Figure 11, temperature is to the graphic representation of PEP activity influence.

Figure 12, the temperature stability graphic representation of PEP enzyme.

Figure 13, the pH beta stability line figure of PEP.

Figure 14, the design of graphics of recombinant plasmid pBL-PEP.

In the present invention, " separation ", " purifying " or " substantially pure " DNA refer to, this DNA or fragment have been arranged in the sequence of its both sides and have separated under natural state, refer to that also this DNA or fragment with under the natural state follow group part of nucleic acid to separate, and separate with the protein of in cell, following it.

In the present invention, term " Punctiform aerogenic monad prolyl endopeptidase (or polypeptide) coded sequence " refer to the encode nucleotide sequence of polypeptide with Punctiform aerogenic monad prolyl endopeptidase activity is such as 1-2073 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1. This degenerate sequence refers to, is arranged in the encoder block 1-2073 position nucleotides of SEQ ID NO.1 sequence, have one or more codons be encoded the degenerate codon of same amino acid replace after and the sequence that produces. Because the degeneracy of codon, thus with SEQ ID NO.1 in 1-2073 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out. This term also comprises can be under middle degree stringent condition, goodly under the height stringent condition, with among the SEQ ID NO.1 from the nucleotide sequence of the nucleotide sequence hybridization of nucleotides 1-2073 position. In addition, this term also comprise with SEQ ID NO.1 in from the homology at least 70% of the nucleotide sequence of nucleotides 1-2073 position, better ground at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.

This term also comprises encoding to have variant form albumen, SEQ ID NO.N sequence with the Punctiform aerogenic monad prolyl endopeptidase identical function. These variant forms comprise (but being not limited to): some (are generally 1-90, better ground 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of nucleotides, and several individual (being generally in 60 of 5 ' and/or 3 ' end interpolation, be in 30, more preferably be in 10 goodly, is in 5 best) nucleotides.

In the present invention, " substantially pure " protein or polypeptide refer to that it accounts at least 20% of the total material of sample at least, better ground at least 50%, more preferably at least 80%, at least 90% (by dry weight or weight in wet base) best. Purity can be measured with any suitable method, as measure the purity of polypeptide with post chromatography, PAGE or HPLC method. Substantially pure polypeptide is substantially free of the component of following it under the natural state.

In the present invention, term " Punctiform aerogenic monad prolyl endopeptidase polypeptide " refers to have the polypeptide of the SEQ ID NO.2 sequence of Punctiform aerogenic monad prolyl endopeptidase activity. This term also comprises having and the variant form Punctiform aerogenic monad prolyl endopeptidase identical function, SEQ ID NO.2 sequence. These variant forms comprise (but being not limited to): some (are generally 1-50, better ground 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and C end and/or one of the terminal interpolation of N or several individual (being generally in 20, be in 10, more preferably be in 5 goodly) amino acid. For example, in the art, when replacing with the close or similar amino acid of performance, usually can not change the function of protein. Again such as, add the functions that or several amino acid also can not change protein usually in that C end and/or N are terminal. This term also comprises active fragment and the reactive derivative of Punctiform aerogenic monad prolyl endopeptidase.

The present invention also comprises the polypeptide variant form of Punctiform aerogenic monad prolyl endopeptidase, and these variant forms comprise: same source sequence, conservative variant, allelic variant, natural mutation, induce mutant, under high or low stringency condition can with the coded albumen of the DNA of Punctiform aerogenic monad prolyl endopeptidase DNA hybridization and the polypeptide or the albumen that utilize the antiserum of anti-Punctiform aerogenic monad prolyl endopeptidase polypeptide to obtain. The present invention also provides other polypeptide, as comprises the fusion albumen of Punctiform aerogenic monad prolyl endopeptidase polypeptide or its fragment. Except the polypeptide of total length almost, the present invention also provides the soluble fragments of Punctiform aerogenic monad prolyl endopeptidase polypeptide. Usually, this fragment has at least about 10 continuous amino acids of Punctiform aerogenic monad prolyl endopeptidase peptide sequence, usually at least about 30 continuous amino acids, at least about 50 continuous amino acids in better ground, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.

Invention also provides the similar thing of Punctiform aerogenic monad prolyl endopeptidase or polypeptide. The difference of these similar things and natural Punctiform aerogenic monad prolyl endopeptidase polypeptide can be the difference on the amino acid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time. These polypeptide comprise hereditary variant natural or that induce. Induce variant to obtain by various technology, as by radiation or be exposed to mutagens and produce at random mutagenesis, also can pass through direct mutagenesis method or the biological technology of other known moleculars. Similar thing can also comprise having the similar thing that is different from the amino acid whose residue of natural L-(such as D-amino acid), and the similar thing with that non-natural exists or synthetic amino acid (such as β, gamma-amino acid). Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.

(usually the not changing a level structure) form of modification comprises: chemically derived form such as acetyl or the carboxylated of the polypeptide that body is interior or external. Modification also comprises the sugar baseization, carries out glycosylation modified and polypeptide generation in the procedure of processing such as those in the synthetic and processing of polypeptide or further. This kind modification can be finished by polypeptide being exposed to the enzyme (such as mammiferous sugar baseization enzyme or deglycosylating enzyme) that carries out the sugar baseization. Modified forms also comprises have the phosphorylated amino acid residue sequence of (such as phosphoric acid tyrosine, phosphoserine, phosphoric acid threonine). Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.

In the present invention, " Punctiform aerogenic monad prolyl endopeptidase conservative variation polypeptide " refers to compare with the amino acid sequence of SEQ ID No. 2, there are 10 at the most, 8 at the most on better ground, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best. These conservative variation polypeptide preferably carry out amino acid substitution according to Table A and produce.

Table A

Initial residue	Representational replacement	The preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala

His(H)	Asn；Gln；Lys；Arg	Arg
			Ile(I)	Leu；Val；Met；Ala；Phe	Leu
Leu(L)	Ile；Val；Met；Ala；Phe	Ile
			Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
			Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
Pro(P)	Ala	Ala
			Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
			Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
			Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The present invention also comprises the antisense sequences of Punctiform aerogenic monad prolyl endopeptidase polypeptid coding sequence and fragment thereof.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of Punctiform aerogenic monad prolyl endopeptidase nucleotide sequence, a better ground 15-50 continuous nucleotide usually. This probe can be used for whether existing in the test sample nucleic acid molecules of peptase in the coding point-like aerogenesis monad prolyl.

The present invention also comprises the method that detects the Punctiform aerogenic monad prolyl endopeptidase nucleotide sequence, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of Punctiform aerogenic monad prolyl endopeptidase polypeptide.Primer length is generally 20-50 Nucleotide.

In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.

As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.

In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is a prokaryotic cell prokaryocyte, more preferably is intestinal bacteria.

In case expressed after the polypeptide of the present invention of reorganization, available various ordinary methods are carried out separation and purification.Be preferably as follows and separate: ultrasonication, ammonium sulfate precipitation and column chromatography.Wherein, more preferably, column chromatography is to carry out with Sephadex G-25, High Performance Q-Sepharose FF, PhenylSepharose 6FF post successively.

On the other hand, the present invention also comprises Punctiform aerogenic monad prolyl endopeptidase DNA or the polypeptide of its fragment coding has specific antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into Punctiform aerogenic monad prolyl endopeptidase gene product or fragment.Preferably, refer to that those can combine with Punctiform aerogenic monad prolyl endopeptidase gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the proteic molecule of Punctiform aerogenic monad prolyl endopeptidase, comprise that also those do not influence the antibody of Punctiform aerogenic monad prolyl endopeptidase protein function.The present invention also comprise those can with modify or without the Punctiform aerogenic monad prolyl endopeptidase gene product bonded antibody of modified forms.

The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab) 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody.

Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the Punctiform aerogenic monad prolyl endopeptidase gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Punctiform aerogenic monad prolyl endopeptidase or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can be utilized hybridoma technology to prepare (to see people such as Kohler, Nature256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the Punctiform aerogenic monad prolyl endopeptidase function and the antibody that does not influence the Punctiform aerogenic monad prolyl endopeptidase function.Each antibody-like of the present invention can utilize the fragment or the functional zone of Punctiform aerogenic monad prolyl endopeptidase gene product, obtains by immunological technique.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of Punctiform aerogenic monad prolyl endopeptidase gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).

As above obtain the inhibitor that these antibody can be used as the novel prolyl endopeptidase of the present invention.

Punctiform aerogenic monad prolyl endopeptidase Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.

For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available point-like aerogenesis aeromonas strain (as point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata)) as template, amplification and must relevant sequence.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally with its human cloning carrier, changes cell over to again, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the original bacterium of selecting for use is to have prolyl endopeptidase (Prolylendopeptidase abbreviates " PEP " as) active point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctatasubsp.punctata).Its chromosomal DNA is reclaimed the dna segment of 8～16Kb with the partially digested back of EcoR I, be connected back Transformed E .coli DH5 α with EcoRI digested vector pUC19, (Benzyloxycarbonyl-Gly-Pro-β-naphthylamide) therefrom screen a strain positive colony obtains the active part of 3.5KbEcoRI/HincII behind the 12Kb EcoRI sheet cracked ends subclone on its recombinant plasmid with the specificity substrate carbobenzoxy-(Cbz) glycyl prolyl-Beta Naphthol 99MIN of this enzyme.Make up this pulsating restriction mapping and carry out the mensuration of nucleotide sequence, obtaining the PEP mrna length is the complete open reading frame of 2073bp, and its amino acid primary structure has very high homology with the PEP that has cloned.Made up the genetic engineering bacterium BL21/ β GEM-PEP that a strain composing type efficiently expresses PEP subsequently.Confirmed the exactness that expression plasmid makes up through dna sequence analysis.Engineering bacteria BL21/pGEM-PEP is deposited in China on November 9th, 1998, the Chinese typical culture collection center (CCTCC) of Wuhan City, and preserving number is CCTCC M98016.

The expression amount of BL21/pGEM-PEP PEP in the YH substratum accounts for about 30% of bacterial protein, and vigor is 112 times of wild bacterium, and expression product is mainly the intracellular protein of solubility, only has a spot of product to be secreted into outside the born of the same parents.Non-reduced SDS-PAGE is shown as monomer, and it is 76464.61Da that electrospray ionization mass spectrum is measured its molecular weight, and is consistent with the molecular weight of gene order prediction.To have obtained purity be 99% reorganization prolyl endopeptidase (PEP) to purifying behind the shake-flask culture, is 67.6U/mg than vigor.Measured 10 aminoacid sequences of N end, with dna sequence dna infer consistent.The iso-electric point of this enzyme is pI=6.05, is 67.6U/mg than vigor, is 0.0311mM to the enzymolysis constant K m of Z-Gly-Pro-β NA substrate.The optimum temperuture of enzyme is that 32 ℃, optimum pH are 8.4, and the thermostability experiment of this proteolytic enzyme shows that stability more stable at 4-32 ℃, the pH value shows that the pH value is more stable at 6-10; The restraining effect result of some proteinase inhibitor and metal ion shows that the PEP enzyme is not suppressed with most of ionic by PMSF, TLCK, TPCK, trypsin inhibitor, pepstatin, EDTA, sodium tetrathionate etc., but obviously be subjected to SDS, Zn ²⁺Inhibition.The enzyme of pitocin 9 peptides and thyrocalcitonin 32 peptides is tested conscientiously and is shown this enzyme hydrolysis proline(Pro) carboxy-terminal peptide bond specifically, but efficient is obviously low than Z-Gly-Pro-β NA.

Engineering strain E.coli BL21/pGEM-PEP can constitutive expression the Punctiform aerogenic monad prolyl endopeptidase of reorganization, but culture condition greatly affects the expression amount and the activity of enzyme, in order to obtain efficiently expressing of this enzyme, the fermentation condition of engineering bacteria is optimized.

Usually, the culture condition of transformed host cells of the present invention is: tryptone 10-20g/L; Yeast extract 5-10g/L KH ₂PO ₄1-4g/L; K ₂HPO ₄2-6g/L; Na ₂HPO ₄12H ₂O5-10g/L; (NH ₄) ₂₈O ₄0.5-2g/L; NH ₄Cl0.1-0.5g/L; MgSO ₄7H ₂O0.5-1g/L.Shaking speed is 100-400rpm, and culture temperature is 28-38 ℃, and medium pH is 6.5-7.2, and incubation time is 12-24h.For example, can select following concrete culture condition for use: tryptone 15g/L; Yeast extract 7.5g/L; KH ₂PO ₄2g/L; K ₂HPO ₄4g/L; Na ₂HPO ₄12H ₂O7g/L; (NH ₄) ₂SO ₄1/2g/L; NH ₄Cl0.2g/L; MgSO ₄7H ₂O1g/L.Shaking speed is 250rpm, and culture temperature is 32 ℃, and medium pH is 7.0, and incubation time is 16h.

The novel prolyl endopeptidase of the present invention has the application with inferior aspect:

(1) as molecular biological reagent enzyme

(2) potential medical applications

People such as Walter in the homogenate of human body uterine cancer cell, found first in 1971 PEP (Walter R, ShlankH, Glass J D et al.Science, 1971,173:827).PEP can effectively degrade in vivo and contain the biologically active peptides of proline(Pro), as (Walter R, Simmons W H, Yoshimoto T.Mol Biochem such as neurotensin, thyrotropic hormone liberin, P material and antidiuretic hormones, 1980,30 (2): 111).Therefore the active ANOMALOUS VARIATIONS of PEP may cause the unusual of the physiological function relevant with these bioactive peptides in the body, even the generation of disease, as finding schizophreniac's blood plasma (Maes M clinically, De Meester I, Scharp S et al.ActaPsychiatr Scand, 1996,93:1), morphine abstinence syndrome disease patient hypothalamus (Juhana J, Pekka R, Raimok etal.Pharmacol ﹠amp; Toxical, 1996,78:129) and in the Alzheimer ' s Disease patient cerebral tissue (AoyagiT, Wasa T, Nagai M et al.Ecperientia, 1990, PEP activity 46:94) all takes place unusually.The specific inhibitor of some PEP (as JTP-4891) is presented at good pharmacological action (the Toide K that improves memory in the mouse memory obstacle model that scopolamine causes, Iwamoto Y, Fujiwara T et al.J Pharmacol Exp Ther1995,274 (3): 1370-1378; Shinoda M, Matsuo A, Toide K.Eur J Pharmacol 1996,305 (1-3): 31-38; Yoshimoto T, Kado K, Matsubara F et al.J Pharmacbiodyn 1987,10:730-735), and in the human body pharmacological evaluation, also obtained ideal results (Umemura K, Kondo K, Ikeda Y et al.Br JClin Pharmacol 1997,43 (6): 613-618).Therefore the further research of PEP and specific inhibitor thereof might be developed the medicine of diseases such as the senile dysmnesia of treatment, senile dementia, dysthymia disorders, schizophrenia.

(3) be applied to polypeptide industry

PEP also can utilize its specific Pro restriction enzyme site except having the potential medical applications, utilize intestinal bacteria tandem expression active polypeptide, and the Pro connection site of design polypeptide prepares active polypeptide in a large number; In addition, utilize the inverse process of this enzyme can catalysis composite reactive polypeptide.Kokubo PEP catalysis pitocin precursor and luteinizing hormone-releasing hormone (LRH) (LHRH) precursor syntocinin and LHRH, yield reaches 55% and 67% respectively, and does not have by product to produce; Therefore, PEP is of great importance in production of polypeptide gene engineering and peptide hormone industrial production.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, such as people such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

Material

(1) bacterial strain and plasmid:

Point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata) are available from Inst. of Hydrobiology, Chinese Academy of Sciences,

Host bacterium E.coli DH5a, E.coli BL2l (DE3) (hsdS gal (λ cIts857 indl Sam7 nin5lacUV5-T7)), plasmid pBluescript SK (+), pUC19, pGEM-3zf are available from Pu Luomaige (Promega) company.

(2) enzyme and reagent:

Molecular cloning toolenzyme and reagent, bacterial genomes, plasmid extraction test kit are Pu Luomaige (Promega) company product;

PMSF, TLCK, TPCK, pepstatin (pepstatin), Trypsin inhibitor SBTI (soybean trypsin inhibitor), sodium tetrathionate (sodium tetrathionate), developer Fast GarnetGBC Salt, pitocin 9 peptides, salmon calcitonin see calcimar 32 peptides are available from Sigma company;

PEP specific substrate benzyloxycarbonyl-Gly-Pro-betanaphthyl acid amides (Benzyloxycarbonyl-Gly-Pro-β-naphthylamide abbreviates " Z-Gly-Pro-β NA " as) is synthetic according to a conventional method;

Purifying protein is a Pharmacia company product with resin Sephadex G-25, Phenyl Sepharose 6FF, High Q-Sepharose FF;

(10mm * 100mm) is a Waters company product to Protein-Pak DEAE 15HR AP-1.

(3) substratum:

The LB substratum, the document Sambrook J that sees reference that fills a prescription, Fristsh E F, Maniatis T. Molecular Cloning:A Laboratory Mannul.2nd ed.NY:Cold Spring Harbor LaboratoryPress, 1989.

The YH substratum contains: peptone 15g/L, yeast powder 7.5g/L, glucose 1g/L, KH ₂PO ₄2g/L, K ₂HPO ₄4g/L, Na ₂HPO ₄12H ₂O7g/L, (NH ₄) ₂SO ₄1.2g/L, NH ₄Cl 0.2g/L, medium pH are 7.0.

Method

The molecular biology working method

The measuring method of PEP enzymic activity: the fermented liquid 10pl that gets the enzyme liquid 10 μ l of dilution or ultrasonication joins and is preheating to 32 ℃, 0.400ml in the damping fluid of the certain pH value of 20mM, the Z-Gly-Pro-β NA solution that adds 10 μ l 5mM behind the insulation 1min at once, mixing timing immediately, reaction 5min, add the Fast Garnet GBC Salt solution colour developing 20min of 250pl 1mg/ml, measure absorbance value in the 550nm place, vigor is represented with optical density(OD) relatively.

The recovery of plasmid extraction, endonuclease digestion, dna segment, connection and transformed into escherichia coli: working method is referring to Sambrook J, Fristsh E F, Maniatis T.Molecular Cloning:A Laboratory Mannul.2nded.NY:Cold Spring Harbor Laboratory Press, 1989, pp.16-340.

The mensuration of expression of recombinant proteins amount: with reference to Sambrook J, Fristsh E F, Maniatis T.MolecularCloning:A Laboratory Mannul.2nd ed.NY:Cold Spring Harbor Laboratory Press, 1989, method is carried out.

The clone of embodiment 1 PEP gene

The structure of chromosome library and the screening of positive colony

With Pu Luomaige test kit Wizard ^Genomic DNA (Promega), the bacterial chromosomal dna of extracting point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata).Reclaim the dna segment of 8～16Kb behind the chromosomal DNA usefulness EcoRI partial digestion that extracts with 0.7% agarose gel electrophoresis, be connected back transformed into escherichia coli E.coli DH5 α with the pUC19 carrier of EcoRI digestion, transformant is coated and is cultivated 16 hours (Fig. 1) on the LB flat board that contains Amp, IPTG and X-Gal.According to develop the color the in vain transformant of picking white of indigo plant, be inoculated in each holes of 96 hole enzyme plates (every hole contains LB180 μ l), 37 ℃ of cultivations are got 50 μ l bacterium liquid after 16～18 hours and are transferred to another 96 orifice plate, the Z-Gly-Pro-β NA solution that adds 50 μ l 5mmol/L, 37 ℃ of reactions add the developer of 50 μ l 1mg/ml after 16～18 hours, what present redness is positive strain.

The evaluation of clone's recombinant plasmid of PEP gene: from constructed chromosome library, screened the recon of about 2000 whites with the activity screen method, wherein a strain presents strong red reaction, and its PEP activity is quite active with the wild point-like aerogenesis of PEP Zymomonas mobilis.The EcoRI that contains the 12Kb that has an appointment in the recombinant plasmid of the called after pAPEP01 of this bacterial strain inserts the segment (see figure 1).

The subclone of PEP gene and the evaluation of recombinant plasmid: reclaim acquisition 4.0Kb, 3.5Kb and three segments of 2.0Kb to cutting the back with the HincII enzyme from the 12Kb EcoRI segment of pAPEP01.They are cut with the plasmid pUC19 of HincII/EcoRI double digestion with the HincII enzyme respectively and are connected transformed competence colibacillus E.coliDH5 α (Fig. 2).The result, in the pulsating connection transformant of 3.5Kb, screening obtains the positive strain that 16 strain PEP activity significantly improve (about 10 times), get the enzyme that wherein four strains carry out recombinant plasmid and cut evaluation, all there is the HincII/EcoRI of an about 3.5Kb to insert segment on the multiple clone site of discovery pUC19, with this recombinant plasmid called after pAPEP11 (see figure 2).

Embodiment 2

The structure of the restriction endonuclease map of point-like aerogenesis Zymomonas mobilis PEP gene and the mensuration of PEP gene nucleotide series:

To the plasmid pAPEP11 that obtains among the embodiment 1, the method that combines with single endonuclease digestion and double digestion has made up PstI, SmaI, XhoI, HindIII and the KpnI zymogram (see figure 3) on the HincII/EcoRI segment.

According to physical map, made up a series of overlapped recombinant plasmids, check order with the Nucleotide automatic sequencer.In the 2.6Kb nucleotide sequence of being measured, relatively find to exist the complete open reading frame (ORF) of PEP gene through homology, the length of PEP ORF is 2073bp.Nucleotide sequence and amino acid sequence coded are respectively SEQ ID No.1 and SEQ ID No.2, are shown in Figure 4 and 5 in addition.

The homology of embodiment 3 point-like aerogenesis Zymomonas mobilis PEP relatively

Adopt PC/GENE software, PEP amino acid primary structure and other aminoacid sequence of report of originating PEP are carried out homology relatively (Fig. 6).

As shown in the figure, point-like aerogenesis Zymomonas mobilis of the present invention is respectively 92.3%, 53.2%, 20.5%, 33.5% and 33.2% with the amino acid identity of A.hydrophlia, the F.meningosepticum, P.furiosus, pig brain and the human lymphocyte PEP that have cloned, at the Ser that forms the catalysis triplet ⁵³⁸, Asp ⁶²²And His ⁶⁵⁷Near have the aminoacid sequence of high conservative, also have the conserved structure of this PEP family of Gly-X-Ser-X-Gly near the Ser of active centre.Therefore having confirmed to separate what obtain is a kind of PEP gene of new source.

The structure of embodiment 4 engineering bacteria BL21/pGEM-PEP

As shown in Figure 7, plasmid pAPEP11 cuts through HincII and EcoRI enzyme, reclaims the dna segment about 3.5kb, is connected on the pGEM-3zf that enzyme is cut, and is built into recombinant plasmid pGEM-PEP (see figure 7),

Recombinant plasmid is transformed among the intestinal bacteria E.coli BL21 again, has made up engineering bacteria BL21/pGEM-PEP, it can efficiently express the PEP of reorganization.This project bacterium BL21/pGEM-PEP is deposited in China on November 9th, 1998, the Chinese typical culture collection center (CCTCC) of Wuhan City, and preserving number is CCTCC M98016.

Ferment on the YH substratum, the result has detected the PEP activity in YH fermented liquid supernatant and born of the same parents, and vigor is 112 times of wild bacterium.The SDS-PAGE electrophoresis showed, PEP mainly is present in the born of the same parents, and the PEP of secretion outside born of the same parents is less than 5%, PEP in the born of the same parents accounts for about 30% of bacterial protein, and do not contain PEP in the centrifugal precipitation behind the broken bacterium, and illustrate that PEP exists with solvable state, do not form inclusion body.

The preparation of embodiment 5 Punctiform aerogenic monad prolyl endopeptidases

The fermentation of engineering bacteria

The fermentation of engineering bacteria is carried out in NBS BioFio 3000 type 5L automatic control jars.Fermented bacterium needs the bacterial strain of the fresh conversion of recombinant plasmid, picking list bacterium colony inserts in the YH test tube 32 ℃ and is cultured to logarithmic phase as first order seed from plate, being linked into the 500ml that contains 100ml YH substratum with 2% inoculum size again shakes in the bottle, 32 ℃, 260rpm cultivates 6h, as secondary seed, in order to last jar.Do not regulate the pH value in the shake-flask culture process.Press 5% of NBS BioFlo 3000 type fermentor tank working volumes and insert secondary seed, cultivate 20hrs for 32 ℃, by adding 25% ammoniacal liquor control pH about 7.0, regulate rotating speed automatically and feed pure oxygen that dissolved oxygen is maintained about 50% in the culturing process.And add feed liquid.The separation and purification of recombinant protein

Bacterial cell disruption: the centrifugal 25min of 5000rpm collects thalline after the fermentation ends, by 10: 1 suspension wet thallus, uses Sonics ﹠amp with PBS in the ice bath; The Materials ultrasonic apparatus is interrupted ultrasonic 20 times, the broken thalline of each 60s, 15, the centrifugal 15min of 000rpm, add ammonium sulfate to 20% saturation ratio in the supernatant, adding ammonium sulfate to 65% saturation ratio in the same centrifugal supernatant, carrying out post after the centrifugal precipitation is dissolved with 20mM Tris-HCl (pH8.0) damping fluid and separate.

Column chromatography for separation: (Φ 2.5 * 30cm) desalinations are collected and are gone up High Q-Sepharose FF behind the sample (Φ 2 * 25cm), and with 0-0.5M NaCl linear gradient elution, corresponding SDS-PAGE electrophoresis is collected the active peak of PEP with Sephadex G-25 after the sample dissolution; The sample of collecting is used 65% ammonium sulfate precipitation again, with 20mM Tris-HCl (pH8.0) damping fluid dissolving back Phenyl Sepharose 6FF (Φ 2 * 10cm) purifying, with 1.5-0M (NR4) 2SO4 linear gradient elution, elution peak is walked the SDS-PAGE electrophoresis, collect PEP, (the HPLC purifying of 10mm * 100mm) is collected the PEP peak, can obtain the pure product of product with Protein-Pak DEAE 15HRAP-1.

The character of embodiment 6 Punctiform aerogenic monad prolyl endopeptidases

For the purifying PEP that obtains among the embodiment 5, the following evaluation and property research.

(a) reorganization PEP is than the mensuration of vigor

β-NA and dyestuff Fast GBC that the specificity substrate Z-G-P-β NA of PEP discharges behind the PEP enzymolysis are formed on the azo compound that 550nm has absorption, A ₅₅₀Can represent the relative vigor of enzyme, corresponding typical curve can calculate the ratio vigor of PEP.The calculation formula of vigor is: than vigor (U/mg)=(479.52A ₅₅₀+ 1.0677)/and C, wherein C represents the concentration (μ g/ml) of enzyme.

Final measurement result, the ratio vigor of PEP is 67.6U/mg.

(b) N of PEP end order-checking

The N end order-checking of PEP entrusts Shanghai Inst. of Biochemistry, Chinese Academy of Sciences to measure, and mass spectrum entrusts Shanghai Organic Chemistry Institute, Chinese Academy of Sciences to measure.Measuring all adopts this area ordinary method to carry out.

As a result, the N terminal amino acid sequence analytical results of PEP is: M-S-G-K-A-R-L-H-Y-P, and with the consensus amino acid sequence (Fig. 5) of DNA supposition.

(c) evaluation of PEP purity

HPLC analyzes and adopts Waters Millennium 2010 C-8 of system reversed-phase columns, second cyanogen 0.1%TFA gradient, and 280nm detects.

As a result, the purity of the Punctiform aerogenic monad prolyl endopeptidase of separation and purification is about 99% among the embodiment 5.

(d) the specific checking of PEP restriction enzyme site

Pitocin 9 peptides and thyrocalcitonin 32 peptides are dissolved in 20mM Tris-Cl (pH8.5) damping fluid, are diluted to 1mg/ml, according to substrate: the amount of enzyme=50: 1 adds the PEP enzyme, and 32 ℃ of enzymes are cut 10hrs.(Waters μ-Bond pak 5 μ 100A 4.6 * 250mm) enzyme analysis in Waters Millennium 2010 systems is cut the peptide spectrum, uses second cyanogen gradient (containing 0.1%TFA), and 210nm detects with the C18 reversed-phase column.

Get enzyme respectively and cut the sample detection of pitocin 4h, the result is a complete degestion, and the point of contact is the Pro site.

Enzyme cuts that sampling detects behind thyrocalcitonin 32 peptide 5h and the 10h respectively, and the result is as follows: the peak of two little peptides that 9 peptides and 23 peptases cut occurred, identified correct through mass spectrum.As seen PEP specificity ground enzyme is cut this site:

(e) molecular weight of Punctiform aerogenic monad prolyl endopeptidase

Measure the molecular weight of reorganization PEP with gel electrophoresis and size-exclusion HPLC.

The molecular weight that the relation that is inversely proportional to according to SDS-PAGE electrophoretic mobility and molecular weight logarithmic value records sample is 76kDa.When HPLC measured the PEP molecular weight, the volume of buffer solution elution and molecular weight logarithmic value were inversely proportional to, and calculation formula is Y=301522e ^-0.1176XWherein Y represents molecular weight (Dal), and X represents elution volume (ml), and the elution volume of PEP is 11.7ml, and then molecular weight is 76kDa (Fig. 8), and the molecular weight that electrospray ionization mass spectrum is measured PEP is 76464.61Da.

(f) mensuration of Punctiform aerogenic monad prolyl endopeptidase iso-electric point

Adopt the miniature polyacrylamide isoelectrofocusing instrument (Mini IEF Cell 111) of Bio-Rad company to measure, electrophoresis method is pressed the Bio-Rad data.Amphotericeledrolyte the pH scope be 3-10.Last sample 2 μ l, electrophoresis finish back Coomassie brilliant blue dyeing, corresponding standard pH analysis of protein iso-electric point.

As a result, the iso-electric point pI=6.05 of Punctiform aerogenic monad prolyl endopeptidase (calculated value is 5.64) sees Fig. 9.

(g) optimum temperuture of Punctiform aerogenic monad prolyl endopeptidase and pH value

With Z-Gly-Pro-β NA is substrate, and the pH scope is active higher between 8-9, and optimal pH is about pH=8.4, and active when pH9.5 also have about 50%, so PEP enzyme vigor better (Figure 10) under alkaline condition.

Optimum temperuture is determined as 30-32 ℃, has kept about 25% vigor at 0 ℃, at 10 ℃ 51% vigor is arranged, and visible PEP has at low temperatures kept higher enzymic activity (Figure 11).

(h) thermostability of Punctiform aerogenic monad prolyl endopeptidase

Be determined at the enzyme liquid of insulation 5h under 20,32,37,50 ℃ of differing tempss, still the situation of Sheng enzyme activity.The result shows that PEP is better in the stability less than 32 ℃, and vigor only remains 36.7% behind 50 ℃ of 1h, shows that simultaneously the thermotolerance of this pure enzyme is bad (Figure 12).Placing the 48h vigor in 4 ℃ refrigerator does not change.

(i) the pH value stabilization of Punctiform aerogenic monad prolyl endopeptidase

Enzyme liquid and different damping fluid behind 26 ℃ of insulation 2h, are surveyed its vigor by the standard enzyme assay method of living, and the enzyme activity that records with insulation under optimal reaction pH is 100%.The result shows this enzyme in the pH6-10 scope relatively stable (Figure 13).

(j) the Km pH-value determination pH of Punctiform aerogenic monad prolyl endopeptidase

The concentration of PEP enzyme is 1 * 10 ^-9Mol/L, concentration of substrate is 10 ^-4-10 ^-5In the M scope, surpass 8 * 10 ^-4M has tangible substrate restraining effect.Reaction times at 5min with interior best.Michaelis-Menton equation according to Michaelis and Menten derivation: v=V[S]/(Km+[S]), conversion obtains: [S]/v=[S]/v+Km/V, obtain the Km value by the Hanes-Woolf graphing method, be 0.0311mM.

(h) inhibition of Punctiform aerogenic monad prolyl endopeptidase

Carry out inhibition test with the inhibitor of a series of proteolytic enzyme is following:,, as above measure residual enzyme work then 32 ℃ of incubations 5 minutes with the 20mM Tris-HCl damping fluid (pH8.4) of Punctiform aerogenic monad prolyl endopeptidase and each inhibitor.The results are shown in shown in table 1 and 2.

Table 1 shows that the PEP enzyme is not subjected to the inhibition of PMSF, TLCK, TPCK, trypsin inhibitor, pepstatin, sodium tetrathionate etc., when PMSF concentration up to 1mM, surpassed 20 times of concentration of substrate, residual activity also has 32.6%.

Table 1

Inhibitor	Concentration	The residue vigor
			Do not have	?0	?100％
?TPCK	?0.1mM	?91.6
			?TPCK	?0.3mM	?52.9
?TLCK	?0.2mM	?88.8
			?TLCK	?1mM	?52.7
Trypsin inhibitor SBTI	?0.1mM	?99.5
			?PMSF	?0.1mM	?91.3
?PMSF	?0.5mM	?61.8

PMSF	1mM	?32.6
			Pepstatin	0.2mM	?60.0
Sodium tetrathionate	0.2mM	?73.5

Table 2 shows, PEP shows the restraining effect of metal ion and other chemical substance and not suppressed by EDTA and most of ionic, but obviously be subjected to SDS, Zn ²⁺Inhibition.

Table 2

Reagent	Concentration (mM)	Remaining activity (%)
			Do not have	?0	?100
Mg 2+	?0.2m	?100
			Zn 2+	?0.2	?5.4
Zn 2+	?0.1	?8.9
			Zn 2+	?0.05	?18.7
Zn 2+	?0.02	?24.1
			Zn 2+	?0.01	?34.5
Fe 2+	?0.2	?95.5
			Cu 2+	?0.2	?61.0
Mn 2+	?0.2	?78
			Ca 2+	?0.2	?98
Co 2+	?0.2	?79.9
			EDTA	?0.2	?109
SDS	?0.1％	?13.4
			SDS	?0.05％	?23.4

Zn ²⁺The activity that obviously suppresses the PEP enzyme.In addition, the PEP enzymic activity does not change with the change of salt ionic concentration (20mM-500mM).

The high-density high expression level fermentation of embodiment 7 recombinant bacterial strain BL21/pGEM-PEP

The fed-batch fermentation technology of optimizing is adopted in the laboratory scale cultivation of 5L, stream adds the less feed liquid of carbonaceous sources, controlling flow acceleration, the dissolved oxygen in the control fermented liquid, the specific growth rate of control engineering bacterium makes the expression of PEP enzyme reach maximum, and the final cell density of fermentation 20hrs reaches 60OD ₆₀₀, be equivalent to dry mycelium 22.5g/L, obtain dry mycelium 98.5g, the expression of enzymes amount is 28%, contains PEP enzyme 3.15g in every liter of fermented liquid.

Then, the PEP enzyme is carried out separation and purification.

The PEP expressed products is a soluble proteins, accounts for about 28% of bacterial protein, therefore can remove the part foreign protein with ammonium sulfate precipitation, can remove a large amount of nucleic acid classes and other non-protein impurity again.From the optimization of different saturation ratio ammonium sulfate as can be seen, PEP mainly is present in the saturation ratio of 20-65%, and the proteic rate of recovery of this step is 36.2%.

The sample maximum volume can arrive 1/10 of resin volume on the Sephadex G-25, and is quicker, thorough than dialysis desalting in a word, avoided causing because of the desalination time is long inactivation and self signs of degradation of enzyme.

Next use the quick captured target albumen of High Q-Sepharose FF, and reach higher separating effect, the purity of enzyme is reached more than 85%.

Phenyl Sepharose 6FF is polishing purification further, and has avoided wanting behind the ammonium sulfate precipitation trouble of desalination, can save time greatly.And the purity purifying of PEP reached 96%, can satisfy requirement fully as the biology tool enzyme.If will reach more highly purified zymin, can be with semipreparative anionic HPLC purifying, one time purifying only needs 30min, can obtain the pure product of nearly 1mg.

The protein recovery of whole purge process is about 10.2%, and activity recovery the results are shown in Table 3 about 27.3%.

Table 3

Program	Protein (mg)	Total activity (U)	Than vigor (U/mg)	Productive rate (%)	The purifying multiple
						Acellular lysate	10,428	?230,000	?22.1	?100	?1.0
Ammonium sulfate precipitation	3,151	?122,000	?38.9	?36.2	?1.2
						Efficient Q sepharose FF	1,482	?90,000	?60.8	?19.0	?3.0
Phenyl sepharose?6FF	860	?56,000	?65.5	?10.2	?3.42

PEP behind the purifying identifies that through SDS-PAGE and HPLC its purity reaches about 99%.

The present invention extensive first and expeditiously purifying reorganization PEP.Ri Ben Akio Kanatani had once reported the purifying of the proline(Pro) end peptidase in Aeromonas hydrophila source on the 113rd phase at J.Biochem.1993 before this, in the 10L fermented liquid only purifying obtained the PEP of 16mg.Therefore, above-mentioned fermentation of the present invention and purifying process are simply quick, the efficient height, be connected between each step rationally, resin adopts the FF type of high flow rate and High Performance type efficiently as far as possible, avoid in the process adopting step consuming time, finish a preparation process and only need 3 days, this proteolytic enzyme for easy inactivation, easy degraded is very favourable.

The preparation of embodiment 8 antibody

Prolyl endopeptidase with the purifying of acquisition among the embodiment 5 is an immunogen, prepares monoclonal antibody by standard method.Found that the antibody that makes can combine with Punctiform aerogenic monad prolyl endopeptidase specifically, and as its inhibitor.

Structure and the expression of embodiment 9 expression plasmid pBL-PEP

Press the identical method of embodiment 4, make up another expression plasmid pBL-PEP, difference only is: use

Plasmid pBL replaces pGEM-3zf, replaces HincII/EcoRI with BamHI/EcoRI when enzyme is cut.The structural diagrams of the recombinant plasmid pBL-PEP that makes up is in Figure 14.

Recombinant plasmid pBL-PEP is transformed among the intestinal bacteria E.coli BL21, has made up engineering bacteria BL21/pBL-PEP, it can efficiently express the PEP of reorganization.In the LB substratum (peptone 10 grams per liters, yeast powder 5 grams per liters, sodium-chlor 10 grams per liters, pH7.0), 30 ℃ are cultured to logarithmic phase (about OD=0.4), change 42 ℃ rapidly over to, temperature-induced 4 hours.Find high expression level PEP but detect, expression amount is 30%.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

(1) general information:

(i) applicant: Shanghai Research Center of Biotechnology

(ii) denomination of invention: Punctiform aerogenic monad prolyl endopeptidase and method for making thereof and purposes

(iii) sequence number: 2

(2) information of SEQ ID NO.1:

(i) sequence signature:

(A) length: 2073bp

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: DNA

( xi ) ：SEQ ID NO.1：ATGTCAGGGA AAGCGCGCCT TCACTACCCC GTCACCCGCC AGGGAGCCCA GGTGGATCAC 60TACTTCGGCC AGGCCGTGGC CGACCCCTAT CGCTGGGTGG AGGATGATCG CAGCCCCGAG 120ACCGAGGCCT GGGTCAAGGC CCAGAATGCC GTGACCCAGG ACTATCTGGC ACAGATCCCG 180TATCGCGCTG CCATCAAGGA GAAGCTGGCC GCCTCCTGGA ACTACGCCAA GGAGGGGGCG 240CCGTTTCGGG AGGGGCGCTA CCACTACTTC TTCAAGAACG ACGGCCTGCA GAACCAGAAC 300GTGCTGTGGC GCCAGAAGGA GGGCAAACCG GCGGAGGTGT TCCTAGATCC CAATACCCTC 360AGCCCCGACG GCACCACGGC GCTGGATCAG CTGAGCTTCT CCCGCGATGG CCGCATCCTG 420GCCTACTCGC TGTCGCTGGC GGGTAGCGAC TGGCGCGAGA TCCACCTGAT GGACGTGGAG 480AGCAAGCAGC CGCTGGAGAC CCCTCTCAAG GACGTGAAAT TCAGCGGCAT CAGCTGGCTC 540GGCAACGAGG GCTTCTTCTA CTCGAGCTAC GACAAGCCCG ATGGCAGCGA GCTGTCGGCC 600AGGACTGATC AGCACAAGGT CTATTTCCAC CGGCTCGGCA CGGCGCAGGA GGATGACCGG 660CTGGTGTTCG GCGCCATCCC GGCCCAGCAC CACCGCTACG TGGGGGCGAC CGTCACCGAA 720GATGACCGCT TCCTGCTCAT CTCGGCGGCG AACTCCACCT CCGGCAACCG CCTCTATGTG 780AAGGATCTGA GCCAGGAGAA CGCGCCGCTG CTGACGGTGC AGGGGGATCT GGACGCGGAC 840GTGAGCCTGG TGGACAACAA GGGCAGCACC CTATACCTGC TGACCAACCG GGACGCCCCC 900AACCGCCGGC TGGTGACGGT GGATGCCGCC AACCCGGGGC CGGCCCACTG GCGCGACCTT 960ATCCCCGAGC GTCACAGGGT GCTGACGGTG CACAGCGGCA GCGGTTATCT GTTCGCCGAG 1020TACATGGTGG ATGCCACCGC CCCGGTCGAG CAGTTCGACT ACGAGGGCAA GCGGGTGCGC 1080GAGGTGGCGC TGCCCGGCCT TGGCAGCGTC AGCGGCTTCA ACGGCAAGCA CGACGACCCC 1140GCCCTCTACT TCGGCTTCGA GAACTATGCC CAGCCGCCCA CTCTCTATCG GTTCGAGCCA 1200AAGAGCGGCG CCATCAGCCT CTATTCCGCC TCGGCGGCGC CGTTCAAGCC GGAGGATTAC 1260GTCTCCGAGC AGCGCTTCTA CCAGAGCAAG GACGGCACCC GGGTGCCGCT CATCATCAGC 1320TACCGCAAGG GGCTGAAACT CGATGGCAGC AACCCGACCA TCCTCTACGG CTATGGCGGT 1380TTTGACGTGA GCCTTACCCC GAGCTTCAGC GTATCGGTGG CCAACTGGCT GGATCTGGGG 1440GGCGTCTATG CGGTGGCCAA CCTGCGTGGG GGCGGCGAGT ACGGCCAGGC CTGGCACCTG 1500GCGGGCACCC AGCAGAACAA ACAGAACGTG TTCGACGACT TCATCGCGGC GGCCGAGTAC 1560CTCAAGGCCG AGGGCTACAC CCGCACCGAT CGGCTGGCGA TCCGCGGTGG CTCCAACGGC 1620GGTCTGCTGG TGGGGGCCGT GATGACCCAG CGGCCGGATC TGATGCGGGT CGCCCTGCCA 1680GCGGTGGGGG TGCTCGACAT GCTGCGCTAC CACACCTTCA CCGCCGGGGC GGGCTGGGCC 1740TATGACTACG GCACCAGTGC CGACAGCGAG GCGATGTTCG ACTACCTGAA GGGCTACTCG 1800CCGCTGCACA ACGTCCGACC CGGGGTCAGC TACCCCTCGA CTATGGTGAC CACGGCGGAT 1860CACGACGATC GGGTGGTACC GGCTCACTCC TTCAAGTTTG CCGCCACCCT GCAGGCCGAC 1920AATGCGGGCC CCCATCCCCA GCTCATCCGC ATCGAGACCA ATGCCGGGCA CGGGGCGGGT 1980ACGCCGGTGG CGAAGCTGAT TGAGCAGAGT GCGGACATCT ATGCCTTCAC CCTGTACGAG 2040ATGGGCTACC GGGAGCTGCC GCGTCAGCCT TGA 2073

(2) information of SEQ ID NO.2:

(i) sequence signature:

(A) length: 690 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: polypeptide

( xi ) ：SEQ ID NO.2：Met Ser Gly Lys Ala Arg Leu His Tyr Pro Val Thr Arg Gln Gly 15Ala Gln Val Asp His Tyr Phe Gly Gln Ala Val Ala Asp Pro Tyr 30Arg Trp Val Glu Asp Asp Arg Ser Pro Glu Thr Glu Ala Trp Val 45Lys Ala Gln Asn Ala Val Thr Gln Asp Tyr Leu Ala Gln Ile Pro 60Tyr Arg Ala Ala Ile Lys Glu Lys Leu Ala Ala Ser Trp Asn Tyr 75Ala Lys Glu Gly Ala Pro Phe Arg Glu Gly Arg Tyr His Tyr Phe 90Phe Lys Asn Asp Gly Leu Gln Asn Gln Asn Val Leu Tyr Arg Gln 105Lys Glu Gly Lys Pro Ala Glu Val Phe Leu Asp Pro Asn Thr Leu 120Ser Pro Asp Gly Thr Thr Ala Leu Asp Gln Leu Ser Phe Ser Arg 135Asp Gly Arg Ile Leu Ala Tyr Ser Leu Ser Leu Ala Gly Ser Asp 150Trp Arg Glu Ile His Leu Met Asp Val Glu Ser Lys Gln Pro Leu 165Glu Thr Pro Leu Lys Asp Val Lys Phe Ser Gly Ile Ser Trp Leu 180Gly Asn Glu Gly Phe Phe Tyr Ser Ser Tyr Asp Lys Pro Asp Gly 195Ser Asp Leu Ser Ala Arg Thr Asp Gln His Lys Val Tyr Phe His 210Arg Leu Gly Thr Ala Gln Glu Asp Asp Arg Leu Val Phe Gly Ala 225Ile Pro Ala Gln His His Arg Tyr Val Gly Ala Thr Val Thr Glu 240Asp Asp Arg Phe Leu Leu Ile Ser Ala Ala Asn Ser Thr Ser Gly 255Asn Arg Leu Tyr Val Lys Asp Leu Ser Gln Glu Asn Ala Pro Leu 270Leu Thr Val Gln Gly Asp Leu Asp Ala Asp Val Ser Leu Val Asp 285Asn Lys Gly Ser Thr Leu Tyr Leu Leu Thr Asn Arg Asp Ala Pro 300Asn Arg Arg Leu Val Thr Val Asp Ala Ala Asn Pro Gly Pro Ala 315His Trp Arg Asp Leu Ile Pro Glu Arg His Arg Val Leu Thr Val 330His Ser Gly Ser Gly Tyr Leu Phe Ala Glu Tyr Met Val Asp Ala 345Thr Ala Pro Val Glu Gln Phe Asp Tyr Glu Gly Lys Arg Val Arg 360Glu Val Ala Leu Pro Gly Leu Gly Ser Val Ser Gly Phe Asn Gly 375Lys His Asp Asp Pro Ala Leu Lys Phe Gly Phe Glu Asn Tyr Ala 390Gln Pro Pro Thr Leu Tyr Arg Phe Glu Pro Lys Ser Gly Ala Ile 405Ser Leu Tyr Ser Ala Ser Ala Ala Pro Phe Lys Pro Glu Asp Tyr 420Val Ser Glu Gln Arg Phe Tyr Gln Ser Lys Asp Gly Thr Arg Val 435Pro Leu Ile Ile Ser Tyr Arg Lys Gly Leu Asp Lys Asp Gly Ser 450Asn Pro Thr Ile Leu Tyr Gly Tyr Gly Gly Phe Asp Val Ser Leu 465Thr Pro Ser Phe Ser Val Ser Val Ala Asn Trp Leu Asp Leu Gly 480Gly Val Tyr Ala Val Ala Asn Leu Arg Gly Gly Gly Glu Tyr Gly 495Gln Ala Trp His Leu Ala Gly Thr Gln Gln Asn Lys Gln Asn Val 510Phe Asp Asp Phe Ile Ala Ala Ala Glu Tyr Leu Lys Ala Glu Gly 525Tyr Thr Arg Thr Asp Arg Leu Ala Ile Arg Gly Gly Ser Asn Gly 540Gly Leu Leu Val Gly Ala Val Met Thr Gln Arg Pro Asp Leu Met 555Arg Val Ala Leu Pro Ala Val Gly Val Leu Asp Met Leu Arg Tyr 570His Tyr Phe Thr Ala Gly Ala Gly Trp Ala Tyr Asp Tyr Gly Thr 585Ser Ala Asp Ser Glu Ala Met Phe Asp Tyr Leu Lys Gly Tyr Ser 600Pro Leu His Met Val Arg Pro Gly Val Ser Tyr Pro Ser Thr Met 615Val Thr Thr Ala Asp His Asp Asp Arg Val Val Pro Ala His Ser 630Phe Lys Phe Ala Ala Thr Leu Gln Ala Asp Asn Ala Gly Pro His 645Pro Gln Leu Ile Arg Ile Glu Thr Asn Ala Gly His Gly Ala Gly 660Thr Pro Val Ala Lys Leu Ile Glu Gln Ser Ala Asp ILe Tyr Ala 675Phe Thr Leu Tyr Glu Met Gly Tyr Arg Glu Leu Pro Arg Gln Pro 690

Claims (10)

1 one kinds of isolating prolyl endopeptidases is characterized in that, this enzyme is the prolyl endopeptidase of point-like aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata), and described endopeptidase is selected from down group:

(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence;

(b) SEQ ID NO:2 aminoacid sequence is formed through disappearance, insertion and/or the replacement of 1-10 amino-acid residue, and have the prolyl endopeptidase enzyme and cut active by (a) polypeptides derived.

2. prolyl endopeptidase according to claim 1 is characterized in that it comprises the aminoacid sequence shown in the SEQ ID No.2.

3. a separated DNA sequence is characterized in that, this dna sequence encoding has the polypeptide of the aminoacid sequence shown in the SEQ ID No.2.

4. dna sequence dna as claimed in claim 3 is characterized in that, this dna sequence dna comprises the nucleotide sequence shown in the 1-2073 position among the SEQ ID No.1.

5. an expression vector is characterized in that, it contains the described dna sequence dna of claim 3.

6. a host cell is characterized in that, it is transformed by the described expression vector of claim 5.

7. host cell as claimed in claim 6 is characterized in that it is e. coli bl21/pGEM-PEP, CCTCC M98016.

8. a method that produces prolyl endopeptidase is characterized in that, this method comprises:

(a) will the encode nucleotide sequence of prolyl endopeptidase of shape point aerogenesis Zymomonas mobilis point-like subspecies (Aeromonas punctata subsp.punctata) operationally is connected in expression regulation sequence, form the prolyl endopeptidase expression vector of some aerogenesis Zymomonas mobilis point-like subspecies, described nucleotide sequence coded polypeptide with the aminoacid sequence shown in the SEQ ID No.2;

(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of prolyl endopeptidase;

(c) be fit to express under the condition of this prolyl endopeptidase polypeptide the reconstitution cell in the culturing step (b);

(d) isolate and have an active polypeptide of prolyl endopeptidase of aerogenesis Zymomonas mobilis point-like subspecies.

9. method as claimed in claim 8 is characterized in that, in step (c), culture condition is: tryptone 10-20g/L; Yeast extract 5-10g/L; KH ₂PO ₄1-4g/L; K ₂HPO ₄2-6g/L; Na ₂HPO ₄12H ₂O 5-10g/L; (NH ₄) ₂SO ₄0.5-2g/L; NH ₄Cl0.1-0.5g/L; MgSO ₄7H ₂O0.5-1g/L, shaking speed is 100-400rpm, and culture temperature is 28-38 ℃, and medium pH is 6.5-7.2, and incubation time is 12-24h.

10. method as claimed in claim 8, it is characterized in that, in step (d), further comprising the steps of: ultrasonication, ammonium sulfate precipitation, carry out column chromatography with Sephadex G-25, High Performance Q-Sepharose FF, Phenyl Sepharose 6FF post then successively.

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