CN101203609B - Production of recombinant proteins by autoproteolytic cleavage of a fusion protein - Google Patents

Abstract

Disclosed is a method for the production of a heterologous polypeptide of interest with a homogenous N-terminus, using a fusion polypeptide comprising the polypeptide of interest and N-terminally thereto a polypeptide exhibiting autoproteolytic function, said method comprising the steps of a) binding of the fusion polypeptide in a soluble, autoproteolytically inactive form by an affinity chromatography system, b) refolding of the fusion polypeptide, thereby activating the autoproteolytic function of the fusion polypeptide and causing cleavage of the heterologous polypeptide of interest, and c) subsequently eluting the heterologous polypeptide of interest, wherein said steps are conducted on one affinity chromatography system.

Description

Shear and prepare recombinant protein by the self-albumen of fusion rotein

Technical field

The present invention relates to a kind of method of preparing the needed heterologous recombination polypeptide (heterologous recombinant polypeptide) with specific homology N end.The present invention combines chromatogram system and fusion polypeptide, this fusion polypeptide comprises needed polypeptide (polypeptide ofinterest) and an extention, have the second polypeptide of self-proteolysis function, this polypeptide is connected with the N end of needed polypeptide.A described chromatogram System forming part of the present invention, in the time that fusion polypeptide is fixed in chromatogram system, this system makes the self-proteolysis function activation of fusion polypeptide N end.Fixing of fusion polypeptide, refolding and shearing are all carried out in same chromatogram system, afterwards, isolate the needed polypeptide of purified form from this chromatogram system.

Background technology

Most of needed polypeptide, for example, come from eukaryotic medicinal use albumen, because it has high expression level speed (expression rate) and high yield (yields), so its preparation is carried out conventionally in bacterial cell.But the mechanism of synthetic polypeptide is different from the mechanism of synthetic polypeptide in eukaryotic cell in bacterial cell; Conventionally, the polypeptide of expressing in bacterial cell all has an additional foreign amino acid at N end, or its N end is (inhomogenous) of homology not, although additional amino acid can be sheared, in most cases this shears and be incomplete.

But especially at field of medicaments, above-mentioned non-homology (inhomogeneity) is unwelcome, because the character that these polypeptide present with natural polypeptides is not identical, for example, the induction of antibody formation, transformation period, pharmacokinetics etc.When natural protein derived N end, and/or N hold not homology N end there will be unacceptable characteristic.When preparation medication polypeptide, require to produce a kind of and natural identical product in most of situation, itself and correct N hold homology,, and there is no plus Amino Acid.Known method is in polypeptide preparation process, to increase some additional steps, attempts whereby to reach above-mentioned requirements, but this method has expended more financial resources and material, and the quite complexity that so-called production downstream process is become.

In bacterial cell, prepare the fusion polypeptide using in the known method of the polypeptide with specific homology N end and include needed polypeptide, and its N end connects a polypeptide with self-proteolytic activity, has the N of the preferred pestivirus of polypeptide of self-proteolytic activity ^prooneself's proteolytic enzyme.Can shear out interested, to there is homology N end polypeptide by the self-hydrolytic activity of fusion polypeptide.

If prepare polypeptide in the tenuigenin of bacterial cell, under certain conditions, the productivity of polypeptide is greater than its folding kinetics (folding kinetics).The polypeptide aggregation body (aggregates) that therefore can form high density, this aggregate is deposited in tenuigenin with the form of inclusion body.In commercial production levels, this to prepare the mode of polypeptide with inclusion body form very interested in above-mentioned for people, because the content of expressed polypeptide in inclusion body is very large, purity is also very high.In addition, the inclusion body in cell lacks proteolytic enzyme, so the polypeptide being stored in inclusion body also can be protected.In addition, inclusion body is highly susceptible to segregation.But the main drawback of preparing polypeptide with tenuigenin inclusion body form is that solubilization of inclusion bodies is very low, and must carry out the refolding of polypeptide.

Therefore, especially, when requiring correct refolding when obtaining possessing bioactive interested polypeptide, it is very complicated that the processing of inclusion body just becomes.Have although utilize the self-proteolytic activity of above-mentioned fusion polypeptide to prepare the polypeptide that homology N holds, the purifying process of required product is still very tediously long, especially in the time that its form with tenuigenin inclusion body is expressed.Treatment process comprises a large amount of cleanings (washing), and refolding is sheared the steps such as purifying and separation.

Therefore, prepare fast, cheaply polypeptide, complicated downstream process will be a huge challenge.In industrial production, this situation is more urgent.Therefore, find a kind of production of simple possible and the technique of purified polypeptide is extremely urgent.

Summary of the invention

In the present invention, the discovery that people are surprised for example, obtains the preparation method of required heterologous polypeptide by special affinity chromatography method is combined to facility greatly with the fusion polypeptide system with self-proteolytic activity from inclusion body.Therefore, this preparation method can be undertaken by a kind of assistance of chromatogram system.

First prepare fusion polypeptide.Fusion polypeptide comprises the polypeptide with self-proteolysis function, is preferably the self-proteolysis function of self-proteolytic enzyme, more preferably the N of pestivirus (Pestivirus) ^prothe self-proteolysis function of oneself's proteolytic enzyme and derivative thereof.The C end of polypeptide has self-proteolysis function, and above-mentioned fusion polypeptide also comprises required heterologous polypeptide.

Fusion polypeptide is to hold under the environment of self-proteolytic activity and prepare in host cell suppressing its N.Prepare fusion polypeptide with the sex change form (denatured form) of tenuigenin inclusion body especially.From cell, these inclusion bodys are emanated out and it is dissolved under the environment that keeps self-proteolytic activity inactivation.Fusion rotein is optionally fixed in chromatogram system, particularly can keep in the chromatographic column of fusion polypeptide N end inactivation and denatured state.

Once fusion polypeptide is fixed in chromatogram system, necessary, loose impurity component will be cleaned out.

In the time only having fusion polypeptide to depart from from chromatogram system, purification system is the environment that activates self-proteolysis function by the environment change from suppressing self-proteolysis function.The change of this environment makes fusion polypeptide recover its structure originally, thereby the self-proteolysis function of N end part is activated, interested polypeptide is sheared out, that last wash-out composition is out purifying, through required polypeptide overlapping, that there is homology N end, and the N of fusion polypeptide end part continues to be fixed in chromatogram system.

Once fusion polypeptide be fixed on chromatogram system in, step 1) clean loose impurity (or pollutent) composition 2) refolding 3) and shear required polypeptide and 4) the required polypeptide of purifying in same chromatogram system.This facilitates operation greatly.Loose impurity component is highly susceptible to cleaning in this system, and fusion polypeptide keeps fixing with the selectivity of chromatogram system simultaneously.

Embodiment

The invention provides a kind of novel method of preparing the needed heterologous polypeptide with homology N end, the method has greatly been simplified and has been obtained the required complicated treating processes of active polypeptide.

Therefore, the present invention relates to a kind of use includes required polypeptide and is connected to required polypeptide N end, the fusion polypeptide preparation with the polypeptide of self-albumen shearing function has the method for the required polypeptide of allos of homology N end, the method comprises the following steps: a) use affinity chromatography system by fusion polypeptide with soluble, the form of oneself's proteolysis functionally inactive is fixed, b) refolding fusion polypeptide, to activate the self-proteolysis function of fusion polypeptide and required heterologous polypeptide sheared, c) the required heterologous polypeptide of wash-out subsequently, wherein said step is all carried out in an affinity chromatography system.

The term implication relating to is herein as described below:

Term " required heterologous polypeptide " (heterologous polypeptide of interest) means a kind of polypeptide not forming by natural self-proteolytic cleavage natural (fusion) polypeptide, required heterologous polypeptide for example has industrial enzyme (technique enzyme) or has therapeutic activity, especially for human treatment's polypeptide.

Term " fusion polypeptide " means the polypeptide that includes two or more polypeptide.Especially, a fusion polypeptide may comprise an affinity labelling, a self-albumen cutting out section, preferred self-proteolytic enzyme and a needed polypeptide.In the present invention, fusion polypeptide comprises needed polypeptide, and has self-albumen shearing function, and N end connects the polypeptide of needed polypeptide.

Term " sex change form " (denatured form) means in the present invention in restructuring preparation process, normally dissolve obtain after inclusion body as product, there is the expression fusion polypeptide of bioinactivation form.

Term " refolding " means dissolving polypeptide and recovers its structure and bioactive mechanism originally, for example, by the protein restructuring in sex change, inactivation form, makes it recover its activity morphology.

Term " self-proteolysis function " (autoproteolytic function) means the self-proteolytic activity that merges a kind of composition in composition (fusion partner), when fusion polypeptide is during in sex change form, this activity inhibited, in the time that fusion polypeptide is carried out refolding, this activity is activated.

In the time of the self-proteolysis functionally inactive of fusion polypeptide, fusion polypeptide is fixed in chromatogram system.In environment change, the shearing of needed polypeptide and process afterwards, fusion polypeptide all must remain fixed in chromatogram system.In the present invention, in the time of fusion polypeptide refolding, shear and start to carry out, now fusion polypeptide returns to activity morphology from inactivation form.The invention provides a kind of affinity chromatography system, make under sex change environment, the N end of fusion polypeptide is fixed in this affinity chromatography system, and in the time of environment change, the fusion composition with self-proteolysis function still remains fixed in affinity chromatography system afterwards.When refolding occurs, polypeptide is still fixed in chromatogram system, so require affine system and methods for refolding not to interfere with each other.This problem is solved in the present invention.

Term used herein " make sex change " and mean the original three-dimensional structure of polypeptide destroyed environment.

In the self-proteolytic activity part of fusion polypeptide, refolding can activate the active of this part and cause the beginning of shearing.Meanwhile, needed polypeptide portion recovers its structure originally, and the needed polypeptide of finally shearing just possesses its form originally, active.After shearing, because the self-proteolytic activity part of fusion polypeptide is still fixed in chromatographic column, and before shearing starts, loose impurity component cleans out from chromatographic column, thus from chromatographic column wash-out out be pure, through the needed polypeptide of refolding.Therefore, do not need to carry out again the two-part separation of fusion polypeptide, shear separation or these postorder work of refolding of agent.

In the present invention, fusion polypeptide is in bacterial host cell, is prepared from initial inactivation form.

In a preferred embodiment of the invention, fusion polypeptide is by recombinant expressed being prepared from of form with inclusion body in bacterial host cell, and the conversion (transform) of the host cell using is that the expression vector of the nucleic acid molecule by containing the fusion polypeptide of encoding is achieved.

Term used herein " inclusion body " means the aggregate that contains heterologous polypeptide in the host cell tenuigenin being present in after conversion.Under the microscope, inclusion body is bright spot shape (brightspots), can obtain inclusion body by isolated cell matter.

Term used herein " host cell of conversion " means the cell that comprises coding heterologous polypeptide carrier.

In order to start the shearing in chromatographic column, need to start most and process that polypeptide has been expressed in host cell in suppress the self-proteolytic activity of fusion polypeptide.The polypeptide of expressing is assembled in the tenuigenin of host cell, especially assembles with inclusion body form, and the need for environment of this process keeps the inactivation form of fusion polypeptide.

The bacterial host cell using in the present invention is, for example gram negative bacterium is as Escherichia, for example intestinal bacteria, or other gram negative bacteriums, for example Rhodopseudomonas, as Pseudomonas aeruginosa, or Bacillaceae (caulobacter sp.), as crescent handle bacillus (Caulobactercrescentus), or gram positive bacterium, as bacillus, especially Bacillus subtilus.Preferably intestinal bacteria are as host cell.

The nucleic acid molecule that the expression vector using in the method for the invention comprises the fusion polypeptide of encoding, it comprises the polypeptide with self-proteolysis function, and its C end is connected with required polypeptide.Shear and carry out at the peptide C end with self-proteolytic activity, to form the homology N end of required polypeptide.

In a preferred embodiment of the invention, the polypeptide that has a self-proteolysis function is self-proteolytic enzyme.

Term used herein " self-proteolytic enzyme " means has self-proteolysis function, can self be sheared to the polypeptide getting off from longer polypeptide portion, preferably neutral protease.Those skilled in the art know the concept of self-proteolytic enzyme, and known have a lot of natural self-proteolytic enzyme systems.Studying more deep self-proteolytic enzyme has, and for example virus protease is grown protein (developmental proteins) (as HetR, Hedgehog protein (its carboxyl terminal proteolytic enzyme), RumA proteolytic enzyme field, UmuD, etc.).

The virus (Flaviviridae) of flaviviridae, comprises that pestivirus (pestiviruses) all has NS3 proteolytic enzyme.Shown by yellow fever virus, 2 type dengue fever viruss and west Nile virus, proteolytic enzyme structural domain is positioned on N end～180 residues of NS3, and it can form (in an apparentintramolecular fashion) be sheared in NS2A/2B and NS2B/NS3 contact are sentenced effect and obvious molecule.By hepatitis C and the sequential analysis of GB virus NS are shown, itself and flavivirus (flavivirus) and pestivirus (pestiviruses) NS3 sequence have close contact.

N holds self-proteolytic enzyme also to there will be in aphthovirus genus (aphthoviruses) [foot and mouth disease virus (FMDV)], and this aphthovirus genus belongs to normal chain (positive strand) RNA viruses of Picornaviridae (Picornaviridae).This proteolytic enzyme is also known as leader protein enzyme (Lpro), and it belongs to the papoid section of L-Cysteine HCL Anhydrous.This proteolytic enzyme is except can be by himself from polypeptide is sheared, and also can cause the proteolysis of the 220-kDa group of eukaryotic initiation factor 4G to be degenerated (degradation) and stop adding the protein synthesis of cap dependency (cap-dependent) host cell.Because micro ribonucleic acid RNA does not add cap, so it is continued translation in the time adding cap in conjunction with (cap-binding) protein complex inactivation.But the virus replication in cell cultures does not need blue tongue virus leader protein enzyme gene.

Other two kinds of self-proteolytic enzyme in picornavirus (picornavirus) section are 2A and 3C, and they are with extremely consistent with chymase serine protease (chymotrypsin-likeserine-proteases).Two kinds of proteolytic enzyme are included in the precursor (precursor) of polyprotein.In plant virus, the brief example of self-proteolysis is; leader protein enzyme in beet yellows virus; this leader protein has the C end structure territory of (conserved) class papoid (papain-like) of unprotected (non-conserved) N end structure territory (RNA amplification function) and protection, and it needs self-proteolysis.

Retrovirus, in for example, Gag-Pol polyprotein in human immunodeficiency virus (HIV-1), also can find self-proteolytic enzyme.This polyprotein comprises one 99 amino acid whose proteolytic enzyme, after the proteolytic enzyme polymerization in itself and another polyprotein, just self can be discharged.

Term " self-proteolytic enzyme " preferentially refers to the N of pestivirus ^prooneself's proteolytic enzyme, comprises its all derivatives with self-proteolytic activity.

In the present invention in more further embodiment, the N that self-proteolytic enzyme is pestivirus ^pro, or it has the derivative of self-hydrolysis function.

Pestivirus is small-sized enveloped virus, and it has a genome of directly serving as mRNA.The proteolytic enzyme of the two-strain coding identifying from pestivirus is respectively N ^prooneself's proteolytic enzyme and serine protease NS3.N ^proproteolytic enzyme is positioned at the N end of polyprotein.N ^profirst protein of composition pestivirus polyprotein, and shear and open with core bag nucleic acid-protein below in self-proteolysis process.Shearing occurs in N ^proafter last amino acid of sequence C ys168.

Pestivirus can form a series of pathogenic agent that comprises that other are viral, typical Pestivirus suis (CSFV), border virus (BDV) and cattle disease viral diarrhea virus (BVDV).

In a preferred embodiment of the invention, pestivirus is selected from CSFV, BDV or BVDV, and wherein CSFV is for most preferably.

In the embodiment being more preferably in the present invention, the N of CSFV ^prothe aminoacid sequence of oneself's proteolytic enzyme is as follows:

SEQ ID NO 1：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGR

VTGSDGKLYHIYVCVDGCILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168)，

Or for thering is the N of self-proteolysis function ^prothe aminoacid sequence of the derivative of oneself's proteolytic enzyme.

See again EMBL database, the number of logging in X87939,1 to 168 amino acids, reads to C extreme direction from N end.

When keeping the activity of required self-proteolytic enzyme, while especially preparing the required required heterologous polypeptide with homology N end, the derivative that the present invention has self-proteolysis function is the N by pestivirus ^prooneself's proteolytic enzyme is by sudden change, and particularly amino acid whose replacement, disappearance, interpolation and/or insertion are derivative.The method of preparing said derivative by sudden change is well known to those skilled in the art.The N of different types of heterologous polypeptide of shearing from fusion polypeptide ^prothe character of oneself's proteolytic enzyme can be adjusted by said mutation.Can design polypeptide in the present invention, make its character not only with improvement compared with neutral protease, and still keep pestivirus N ^proself-proteolytic activity.For this consideration, preferably those have improved properties and can preferentially be fixed on the derivative in affinity chromatography system aspect solvability, and these character use in this patent is very big.

The self-proteolysis function of the derivative being obtained by sudden change can be tested by for example WO01/11056.

Preferably cysteine residues is replaced, and aminoacid sequence is the natural pestivirus N described in above-mentioned ID NO 1 ^proderivative.More preferably C112, C134, tri-cysteine residues of C138 for example, are replaced by other amino-acid residues (L-glutamic acid), natural N ^proderivative.Especially preferably there is the derivative of following amino acid sequences:

SEQ ID NO 2：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFEEVTKRIGR

VTGSDGKLYHIYVEVDGEILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168)。

Another preferred natural pestivirus N ^proderivative, except the sudden change of above-mentioned halfcystine, the arginine on 53 and 57 is replaced by glutaminic acid residue, the glycine on 54 is replaced by aspartic acid, the leucin on 143 is replaced by glutamine.The aminoacid sequence of this derivative is as follows:

SEQ ID NO 3：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFEEVTKRIGR

VTGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWIRNFTNCPLWVTSC-(168)。

Therefore on the other hand, the present invention also relates to aforesaid method, the N that wherein fusion polypeptide comprises CSFV ^prothe derivative of oneself's proteolytic enzyme, this derivative, except at least one cysteine residues is replaced as mentioned above, also has at least one hydrophobic amino acid residue to be replaced by hydrophilic residue.

In the present invention, the N of preferred a kind of CSFV ^prothe derivative of oneself's proteolytic enzyme, wherein, except at least one halfcystine is replaced as mentioned above, in following amino acid, at least one of them is also replaced: V24, A27, L32, G54, L75, A109, V114, V121, L143, I155 and F158.Preferred following amino acid is replaced the derivative being formed by Threonine (T): L-Ala (A) 109, α-amino-isovaleric acid (V) 114, Isoleucine (I) 155 and phenylalanine (F) 158.

Therefore on the other hand, the present invention preferentially relates to aforesaid method, the N that wherein fusion polypeptide comprises CSFV ^prothe derivative of oneself's proteolytic enzyme, wherein except at least one cysteine residues described above is replaced, following amino acid is also replaced by Threonine (T): L-Ala (A) 109, α-amino-isovaleric acid (V) 114, Isoleucine (I) 155 and phenylalanine (F) 158.In addition, more preferably there is in the present invention the N of the CSFV of following amino acid sequences ^prothe derivative of oneself's proteolytic enzyme:

SEQ ID NO 4：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV

TGSDGKLYHIYVEVDGEILLKLAKRGTPRTLKWTRNTTNCPLWVTSC-(168)。

Therefore, on the other hand, the present invention more preferentially relates to aforesaid method, and wherein fusion polypeptide comprises the N with the CSFV of aminoacid sequence shown in SEQ ID NO 4 ^prothe derivative of oneself's proteolytic enzyme.

In the present invention, the more preferably N of a kind of CSFV ^prothe derivative of oneself's proteolytic enzyme, this derivative is except at least one halfcystine is replaced as mentioned above, following amino acid: L-Ala (A) 109, α-amino-isovaleric acid (V) 114, Isoleucine (I) 155, phenylalanine (F) 158 is replaced by Threonine (T); Arginine (R) 53 is replaced by L-glutamic acid (E), glycine (G) 54 is replaced by aspartic acid (D), arginine (R) 57 is replaced by L-glutamic acid (E), and leucine (L) 143 is replaced by glutamine (Q).

In the present invention, the N of most preferred CSFV ^prothe derivative of oneself's proteolytic enzyme has following amino acid sequences:

SEQ ID NO 5：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV

TGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWTRNTTNCPLWVTSC-(168)。

Therefore, another most preferred aspect, the present invention relates to aforesaid method equally, wherein fusion polypeptide is containing the N of the CSFV shown in sequence SEQ ID NO 5 ^prothe derivative of oneself's proteolytic enzyme.

Another same preferred aspect, the present invention relates to the above-mentioned method of preparing heterologous protein, the N that wherein fusion polypeptide contains CSFV ^prothe derivative of oneself's proteolytic enzyme, the aminoacid sequence of this derivative is SEQ ID NO 5, wherein asparagine (N) 35 is replaced with to Threonine (T), and Threonine (T) 158 is replaced with to Serine (S).

The N of the CSFV using in method aspect the present invention is above-mentioned ^prothe derivative of oneself's proteolytic enzyme is also a part of the present invention, and its aminoacid sequence is as follows:

SEQ ID NO 50：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGTPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV

TGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWTRNSTNCPLWVTSC-(168)。

Another preferred aspect, the present invention relates to the above-mentioned method of preparing heterologous protein (heterologousprotein), the N that wherein fusion polypeptide contains CSFV ^prothe derivative of oneself's proteolytic enzyme, the aminoacid sequence of this derivative is SEQ ID NO 32, wherein L-Ala (A) 28 is replaced with to L-glutamic acid (E), Serine (S) 71 is replaced with to phenylalanine (F), arginine (R) 150 is replaced with to Histidine (H).

The N of the CSFV using in method aspect the present invention is above-mentioned ^prothe derivative of oneself's proteolytic enzyme is also a part of the present invention, and its aminoacid sequence is as follows:

SEQ ID NO 51：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTEGRPLFGTPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRFGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV

TGSDGKLYHIYVEVDGEILLKQAKRGTPHTLKWTRNSTNCPLWVTSC-(168)。

When preparation proinsulin, in method of the present invention, be the N of the CSFV of SEQ ID NO 32 by sequence ^prothe derivative of oneself's proteolytic enzyme with at least there is proinsulin, preferably with proinsulin, more preferably former with human insulin, most preferably merge with the amino acid whose protein of recombinant human proinsulin front three.

According to the present invention, the N of preferred CSFV ^prothe derivative of oneself's proteolytic enzyme is except at least one cysteine residues is replaced as mentioned above, at least one of them is also replaced following amino acid: arginine (R) 53, glycosyl acetic acid (G) 54, arginine (R) 57, Threonine (T) 109,114,155,158, leucine (L) 143.According to the present invention, the N of preferred CSFV ^prothe derivative of oneself's proteolytic enzyme is except at least one cysteine residues is replaced as mentioned above, following amino acid is replaced by respectively: arginine (R) 53 is replaced by L-glutamic acid (E), glycosyl acetic acid (G) 54 is replaced by aspartic acid (D), arginine (R) 57 is replaced by L-glutamic acid (E), Threonine (T) 109, 114, 155, 158 are replaced by Serine (S), leucine (L) is replaced by glutamine (Q) or asparagine (N) or aspartic acid (D) or Serine (S) or Histidine.

The N of the CSFV preferably using in method aspect the present invention is above-mentioned ^prothe derivative of oneself's proteolytic enzyme is also a part of the present invention, and its aminoacid sequence is as follows:

SEQ ID 52：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDESQFEESTKRIGR

VTGSDGKLYHIYVEVDGEILLKSAKRGTPRTLKWSRNSTNCPLWVTSC-(168)。

SEQ ID 53：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDESQFEESTKRIGR

VTGSDGKLYHIYVEVDGEILLKNAKRGTPRTLKWSRNSTNCPLWVTSC-(168)。

SEQ ID 54：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDESQFEESTKRIGR

VTGSDGKLYHIYVEVDGEILLKDAKRGTPRTLKWSRNSTNCPLWVTSC-(168)。

SEQ ID 55：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDESQFEESTKRIGR

VTGSDGKLYHIYVEVDGEILLKHAKRGTPRTLKWSRNSTNCPLWVTSC-(168)

SEQ ID 56：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT

LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDESQFEESTKRIGC

VTGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWSRNSTNCPLWVTSC-(168)

Expression vector codes is as the needed polypeptide of a part for fusion polypeptide, and it can shear by self-proteolysis.In the present invention, can use above-mentioned expression vector to prepare a series of needed polypeptide.For example, possess the polypeptide of pharmacologically active, it is selected from Interferon, rabbit, interleukin, HGH, somatomedin (growth factor), cytokine, enzyme, enzyme inhibitors, antibody and antibody fragment, like that, for example interferon alpha 2A, interferon alpha 2B, interleukin-3, interleukin-6, human growth hormone, Regular Insulin (former), rhIGF-1, granulocyte colony-stimulating factor, the huge G CFS of biting of granulocyte, macrophage colony stimulating factor, interferon beta 1, ox somatropin (bovine somatropin), pig somatropin (porcine somatropin), interleukin 11, interleukin-2, Fab (Fab-fragment), and little peptide is as thyrocalcitonin, parathyroid gland (PTH), or glucagon, CD40 aglucon soluble form, plasminogen activator, sex steroid is in conjunction with albumen, Urogastron and tissue factor cell foreign lands (tissue factor extracellulardomain).

In addition, required polypeptide can be also the polypeptide of other arbitrary forms, is especially particularly suitable for the polypeptide of analytical procedure, for example egfp (GFP).

In the expression vector using in method of the present invention, fusion polypeptide need operationally be connected with at least one expression control sequenc (expression control sequence).Expression control sequenc is especially, promotor (as lac, tac, T3, T7, trp, gac, vhb, lambda pL or phoA promotor), ribosome binding site (as belonging to the natural ribosome binding site of above-mentioned promotor, cro or synthetic ribosome binding site), or transcription pausing (as rrnB T1T2 or bla).

Carrier also may comprise the sequence of the Fusion domain of encoding, as described below, it is positioned at the N end of fusion polypeptide, and require it to be fixed in affinity chromatography system, for example, polyamino acid is as polylysin, or for so-called " epitope mark (epitopetags) " of immune affinity chromatographic, the known epitope mark that can be used for special monoclonal antibody comprises FLAG, influenza virus hemagglutinin (HA) and proto-oncogene mark.

In a preferred embodiment of the invention, expression vector is plasmid.

The namely expression strain (expression strain) of bacterial host cell transforming, its cultivation is to carry out according to known microbial process (microbiological practice).

Host strain (Host strain) is cultivated and is formed by simple biological group on nutrition base, can certainly use the cell suspending liquid (cell bank) of cryopreservation.Conventionally need to cultivate bacterial strain through multi-stage process and obtain enough biomasss for future use.

In small-scale preparation situation, can be in shaking flask, in most of situation, use complex medium (for example LB broth culture) to cultivate.Also can use the substratum (for example citrate medium) of definite ingredients.Because fusion polypeptide is in a preferred embodiment of the invention the form of insoluble inclusion body, need under the condition of relatively-high temperature, carry out (for example 30 ℃ or 37 ℃) so cultivate.Inducible system is highly suitable for preparing inclusion body (for example containing trp, lac, tac, phoA promotor).

In extensive preparation situation, multilevel hierarchy is made up of a large amount of bio-reactor (fermentor tank), preferentially uses the substratum of definite ingredients.In addition, can also greatly increase biomass and turnout by add special nutrition (adding) in substratum base in batches.In addition, this method is similar with use shake-flask culture.

In the method for the invention, inclusion body is separated from host cell in a kind of known mode.

For example, after fermentation starts, obtain host cell by the combination of centrifuging, micro-filtration method, flocculence or this several method, preferably use centrifuging.Moist cell mass is able to fragmentation by the method for machinery, chemistry or physics, for example high pressure homogenisers, the sand machine that rubs, French cell press, crushing apparatus, osmotic shock method, stain remover, the combination of enzymolysis or above-mentioned several method.Preferably use high pressure homogenisers to make cell fission.In the preferred embodiment of assembling with the form of inclusion body at recombinant fusion polypeptide, can or be more preferably slow-revving simple centrifuging by for example high pressure disperse and obtain inclusion body.By centrifuging or micro-filtration or both are in conjunction with inclusion body being separated.Can by inclusion body being carried out multistage resuspension and improved the purity of required polypeptide in a series of damping fluid, for example, use NaCl (for example 0.5-1.0M) and/or stain remover (for example Triton X100).Preferably carry out multistage cleaning and improve the purity (for example first use 0.5% Septochol, use afterwards the NaCl solution of twice 1M, finally use distilled water) of inclusion body with a series of damping fluid.So just most of impurity polypeptide can be cleared out from inclusion body.

In the time preparing affinity chromatography, need to dissolve the inclusion body of separating.

The present invention relates to aforesaid method, the preferential chromatogram system that uses in this method, the fusion polypeptide preparing is high from liquid sequence and suppress self-proteolysis and live under the environment of row and dissolve.

Term used herein " high from liquid sequence " (high density is from liquid) (chaotropic) means the environment that can not or almost can not observe intramolecular action.Can form this environment by the method that for example adds stain remover, alcohol, urea or guanidine hydrochloric acid.Being applicable to the not above-mentioned environment of homopolypeptide is also not quite similar.The above-mentioned environment that how to confirm is applicable to each peptide species belongs to those skilled in the art's programme area.

Use high density chaotropic agent (chaotropic agent) by solubilization of inclusion bodies.After solubilization of inclusion bodies, can obtain one and reduce in fact in molecule and the unit molecule suspension of intermolecular interaction.Preferred solvent is urea, guanidine hydrochloric acid and strong ion stain remover, for example N-Sarkosyl L (N-lauroylsarcosine).In another preferred embodiment of the present invention, also can use pH value to be alkaline aqueous alcoholic liquid, or pH value is the alkaline aqueous solution and dissolve inclusion body.

Term used herein " dissolving " means the method for dissolving inclusion body.After dissolving, can obtain one has in molecule and the polypeptide unit molecule suspension of intermolecular least action.，

In the present invention, when existing oxidation when cysteine residues, preferably use pH value be 7.3 contain 50mM Tris/HCl, 8M urea, is added with the suspension dissolving inclusion body of for example 50mM DTT of reductive agent.

Necessary time, can pass through for example centrifugal separation remove portion insoluble substance.

After the fusion polypeptide of inactivation prepares with soluble state in cell, the cell homogenates of clarification is carried out to the inclusion body of foregoing operation to obtain being dissolved out.

The polypeptide dissolving is further diluted, and enters afterwards chromatogram system and is fixed on affinity chromatographic column.In the present invention, can regulate chromatogram system, thereby can optionally identify the fusion polypeptide part with self-proteolysis function, and in sex change, chaotropic environment, be fixed in chromatographic column making.In above-mentioned environment, fusion polypeptide sex change inactivation.In the time that fusion polypeptide is fixed in chromatographic column, be the environment that makes renaturation, cosmotropic by making inactivation, chaotropic environment change, fusion polypeptide just can be recovered its structure originally, and reactivates self-proteolysis function.In the process of this environment change, the part with self-proteolysis function is still fixed in chromatographic column.

Term used herein " cosmotropic " means and promotes molecular interaction the environment that impels biological structure to form.The above-mentioned environment that is applicable to differing molecular is also not quite similar.As Citrate trianion, the vitriol and the strongest as the cosmotropic effect of cationic quaternary amine or ammonium of negatively charged ion.Some other reagent, as stain remover or redox system also can be used for promoting the carrying out of refolding.The above-mentioned environment that how to confirm is applicable to each peptide species belongs to those skilled in the art's programme area.

In framework of the present invention, all chromatogram systems can optionally be fixed fusion polypeptide under chaotropic environment (chaotropic condition) in theory, and fix with its maintenance lower continuation of cosmotropiccondition (environment).In a preferred embodiment, the matrix of chromatogram system is chromatographic column form, also can select other forms of matrix, as chromatogram grain or organic substance, and the polyethylene glycol of for example revising with affinity peptide.

Be applicable to chromatogram system of the present invention based on cellulose binding domain (cellulose bindingdomain), can be to use the such as cation-exchange chromatography system of the polycation mark of poly arginine or poly-lysine etc., can be also the anion-exchange chromatography that uses the polyanion marks such as such as poly asparagine.

In the present invention, affinity chromatography system preference is selected from fixing metal ions chromatogram (IMAC), cation-exchange chromatography, anion-exchange chromatography, cellulose binding domain chromatogram and peptide affinity chromatography.

The affinity chromatography system preference cation-exchange chromatography using, in this chromatogram, fusion polypeptide contains polycation mark (polycationic tag), more preferably uses poly arginine or polylysine as affinity labelling.

For cation-exchange chromatography system, the fusion polypeptide of expression comprises N end polycation mark, for example poly arginine or polylysine mark.The solution of the fusion polypeptide that comprises the expression extracting from host cell, (after filtration) is loaded in chromatographic column, is filled with the medium that is applicable to cation-exchange chromatography in chromatographic column, for example SP gel FF, CM gel FF, Fractogel EMD SO ^3-.Preferably use the lower damping fluid of conductivity.After loading well, by loose material wash-out out, use afterwards the damping fluid that urea content is lower to carry out refolding.When urea content is during lower than 0.5M, target protein be sheared and elution from chromatographic column (eluted) out.

In another preferred embodiment of the present invention, also can select anion-exchange chromatography system, in this chromatogram system, fusion polypeptide need contain polyanion mark.Preferably poly asparagine is as affinity labelling.

More in preferred embodiment, also can use fixing metal ions affinity chromatography system (IMAC) to reach above-mentioned fixed characteristic at one.

In a preferred embodiment of the invention, select fixing metal ions affinity chromatography (IMAC) as affinity chromatography system, fusion polypeptide comprises the affine mark of metal-chelating.

In these cases, fusion polypeptide is identified, and is fixed in chromatogram system by the affine mark of its metal-chelating.

At one more in preferred embodiment of the present invention, metal-chelating is affine is labeled as polyhistidyl affinity labelling.

IMAC is for example, based on Histidine or other special acids (amino acid on natural protein surface or the amino acid obtaining by DNA recombinant technology) and various fixing metal ions, copper, nickel, the covalent attachment of zinc or iron ion.The known chromatographic material that is applicable to IMAC (chromatographic materials) is equally applicable in the present invention.In a preferred embodiment of the invention, use Ni ²⁺-Chelating Sepharose Fast flow (GEHealthcare, Uppsala, SE) is as matrix (matrix).

In addition, also can select immune affinity chromatographic, use the fusion polypeptide of N end with above-mentioned epitope mark, be fixed on chromatography matrix by function described in the antibody recognition of this mark.

In addition, in the present invention, use the affinity chromatography system of oligopeptides aglucon also to there is required fixed characteristic.

Term used herein " oligopeptides " (oligopeptides) means and at least comprises three amino acid whose protein compounds.This oligopeptides length is no more than 35 amino acid whose length conventionally.

In a preferred embodiment of the invention, in affinity chromatography system, use the oligopeptides aglucon of 5 to 12 amino acid lengths, more preferably 6 to 8 amino acid lengths and the oligopeptides aglucon that comprises a tryptophan residue, under chaotropic environment, the part that oligopeptides aglucon optionally has a self-proteolysis function in fusion polypeptide is combined, in the time that environmental change is cosmotropic environment, oligopeptides aglucon still keeps combination with the part with self-proteolysis function.

For example from antibody, learn, in the affinity chromatography of this form, use the special combination between some polypeptide and other polypeptide.Oligopeptides also can be used as affinity ligand.These molecules have higher chemical stability, effect and selectivity, and have lower price, and are generally avirulent.These characteristics are very large advantages in bio-pharmaceuticals technique.Those skilled in the art will know that and how from combined peptide library or biological storehouse, to find the directly oligopeptides aglucon for target molecule.According to the present invention, the screening of oligopeptides aglucon need to be carried out in chaotropic environment.

The method of the known synthetic peptide of prior art can be for the preparation of oligopeptides aglucon required in the present invention.The people such as preferred use SPOT synthesis method, PIN synthesis method, teabag synthesis method, Ruiwu Liu are the people such as mix and split method, Joseph A.Buettner described in Experimental Hematology 31 (2003) 11-30 at Int.J.Peptide Protein Res.47 (1996), and the PELICAN legal system described in 70-83 is for oligopeptides aglucon.Can use some to connect chemicals (linkerchemistries) and fix (anchoring) first amino acid.In a preferred embodiment of the invention, can prepare respectively aglucon, then it is fixed on chromatography matrix one by one.In another preferred embodiment of the present invention, can peptide aglucon is directly synthetic on chromatography matrix.

Oligopeptides aglucon has high degree of specificity.In the present invention, the characteristic of the oligopeptides of synthesized is, makes under sex change environment, and this oligopeptides can be optionally in conjunction with N ^pro, N ^proderivative and fusion polypeptide thereof.In the present invention, above-mentioned oligopeptides aglucon is directly for the fusion polypeptide part with autoproteolytic cleavage function.

In a preferred embodiment of the invention, oligopeptides aglucon has and is selected from following aminoacid sequence:

SEQ ID NO 6：VSIFEW，

SEQ IS NO 7：AVSIEWY，

SEQ ID NO 8：AVSFIWY，

SEQ ID NO 9：VSFIWYK，

SEQ ID NO 10：ASRFWYA，

SEQ ID NO 11：AFYTWYA，

SEQ ID NO 12：AFYRWYK，

SEQ ID NO 13：AFYRWY，

SEQ ID NO 14：AFYRWYA，

SEQ ID NO 15：AVSIFEWY，

SEQ ID NO 18：AVSRNWY，

SEQ ID NO 17：ASRFWY，

SEQ ID NO 18：AFYRWYAA，

SEQ ID NO 19：AFYRWY，

SEQ ID NO 20：ASRFWYAA，

SEQ ID NO 21：AFYRWYAA，

SEQ ID NO 22：AFYSWYAA。

In the present invention, can use the oligopeptides aglucon that there is the oligopeptides aglucon of free N end or there is sealing N end, be formed the N end of sealing by for example acetylizing.

In most preferred embodiment of the present invention, use the N that aminoacid sequence is the natural CSFV of SEQ ID NO 5 ^pro, be combined with aminoacid sequence and be selected from the oligopeptides aglucon of SEQ ID NO 10, SEQID NO 11, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14.

Therefore, the present invention also provides the affinity matrix (affinity matrix) that contains solid phase and the affinity ligand that contains the peptide bond that is coupled at this solid phase, and the affinity ligand that wherein contains peptide bond is selected from following aglucon:

A) containing general formula is X ₁x ₂x ₃x ₄peptide, wherein X ₁to X ₄for amino-acid residue, X ₁to X ₄in to have two residues at least be W, Y or F;

B) containing general formula is X ₅x ₆x ₇x ₈peptide, wherein X ₅to X ₈for amino-acid residue, X ₅to X ₈in to have a residue at least be W, X ₅to X ₈in to have a residue at least be E or D; And

C) polyamino acid that the amino acid monomer being made up of R, K, E and D and the amino acid monomer being made up of Y, F and W form, preferably poly-KY, poly-KF, poly-KW, poly-RY, poly-RF, poly-RW, poly-EY, poly-DY, poly-EF, poly-EW, poly-DF and poly-DW.

Condition is that depsipeptides length a) and b) mostly is 35 amino-acid residues most, and the peptide length c) minimum be 20 amino-acid residues.

These affinity ligands are to described self-protease molecule, especially to N ^pro, N ^proderivative and the combination of fusion rotein there is very high affinity.Especially, no matter that above-mentioned aglucon or affinity matrix can fix N under chaotropic environment or under the environment of cosmotropic ^pro, N ^proderivative and fusion rotein thereof, can fix at least the N of for example fusion rotein ^pro.

The length of the peptide (also can be called " oligopeptides " here) a) and b) is preferably 5 to 12 amino-acid residues, more preferably 6 to 8 amino-acid residues.In oligopeptides, preferably has a positively charged amino acid at least.C) preferably at least 35 amino-acid residues of the polyamino acid length shown in, more preferably at least 50 amino-acid residues, most preferably at least 100 amino-acid residues.Preferred polyamino acid is: be for example used as the commercial polyamino acid of substratum, as poly-KW, 4: 1 (MW20.000-50.000 Da; SIGMA product No.P9285), poly-KY, 4: 1 (MW20.000-50.000 Da; SIGMA product No.P4695) or poly-KF, 1: 1 (MW20.000-50.000 Da; SIGMA product No.P3150).

In the present invention, related affinity ligand need to pass through chemically modified, particularly acetylize, esterification, amidation, oxidation, reduction or prepare by connecting molecule.

Preferably by covalent linkage, affinity ligand is connected with solid-phase matrix.

The substrate material having come into operation is at present all suitable to solid-phase matrix.Preferably solid-phase matrix, it is selected from chromatographic material, especially the carrier (support) based on Mierocrystalline cellulose, agarose, acrylamide, polystyrene divinylbenzene (poly (styrene-divinylbenzene)) or vinyl ethylene glycol methacrylic acid ester (ethylene glycol-methacrylate copolymers), microtiter plate, nitrocellulose membrane, microchip, slide or metallic coating carrier.

In the present invention, use the solid phase carrier of a series of kinds, for example, based on Mierocrystalline cellulose, agarose (sepharose or Macro-Prep gel), dextran (sephadex), acrylamide (Sephacryl, Trisacryl gels), anhydrous silicic acid (TSK, SW gel), polystyrene divinylbenzene (poly (styrene divinylbenzene)) (Source or Poros gel), vinyl ethylene glycol methacrylic acid ester (Toyopearl HW, TSK, PW, fractogelEMD gel) or the carrier of above substance mixture, especially the carrier based on agarose and dextran (Superdex gel).Preferably verify by authoritative institution of the U.S. (FDA food and drug administration) or office of European Union, can be used for the carrier of the mankind or livestock.In addition, selected carrier must with the present invention in affinity ligand fix, preferably by covalently bound mode (carrier is wanted functionalization).As the main component of matrix, solid-phase matrix can comprise at present known, be suitable for the material separating with protein or other biological molecule solid phase, these materials can be natural also can synthesizing, can be can be organically also inorganic, for example, natural or synthetic saccharan, as agar and agarose; Mierocrystalline cellulose, ether of cellulose, as hydroxypropylcellulose, carboxymethyl cellulose; Starch; Resin is as guar gum, gum arabic, ghatti gum, tragacanth, Viscogum BE, xanthan gum; Pectin; Saliva Orthana; Dextran; Chitin; Chitosan; Alginate; Angle fork (dish) glue; Vitrum AB; Gelatinum; And synthetic polymer, for example polymeric amide is as polyacrylamide and PMAm; Polyimide; Polyester; Polyethers; Polyvinyl compound, as polyvinyl alcohol and polystyrene; Polyolefine; Inorganic materials, if tripoli material is as silicon-dioxide, comprises soft silica and quartz; Silica; Pure Silicon Metal, controlled pore glass (controlled pore glasses) and pottery; Metal oxide and sulfide, and the combination of the material of above-mentioned natural or synthetic or organic or inorganic.

The main component (backbone) of matrix is agar preferably, agarose, and Mierocrystalline cellulose, ether of cellulose is hydroxypropylcellulose such as, carboxymethyl cellulose, polymeric amide is polyacrylamide such as, polyvinyl alcohol, silica and controlled pore glass.

As the main component of matrix, especially attractive solid phase material has for example agar or the sugared bead of agar (gathering), as the Sepharose bead of Pharmacia Biotech company of Sweden and Superose bead, the Biogel A of Biorad company of the U.S.; Dextran grain is as the ephadex of PharmaciaBiotech company; Mierocrystalline cellulose bead and cellulose membrane are as the Perioza Mierocrystalline cellulose of Secheza company of Czechoslovakia; Compound bead, as Sephacryl and the Superdex of Pharmacia Biotech company; Synthetic organic polymer bead is as the Fractogel of Toso-Haas company of the U.S.; The POROS medium of Perceptive Biosystem company of the U.S., the Bio-Rex of Biorad company, Bio-Gel P and Marcro Prep, the HEMA of TESSEK company and Separon, the Hyper D of BioSepra company of the U.S. and Trisacryl medium, the Enzacryl of Minnesota Mining and Manufacturing Company and Azlactone; Silica grain is as controlled pore glass and the PROSEP of Bioprocesing company of Britain, the Spherocil of BioSepra company; And granular or membranaceous coating silica synthetics, as the ACTI-DISK of Arbor Technologies company of the U.S., ACTI-MOD and CycloSep.

In typical case, solid-phase matrix main component and the solid-phase matrix with special function can be for example irregular particle shapes, spherical shape, membranaceous or sheet, molded surface or bar-shaped.Solid phase material can be designed to protein portion infiltration or infiltration or impermeable completely.In an attractive especially embodiment of the present invention, matrix is irregular particle shape or spherical shape, size is between 1-10000 μ m, preferably between 10-1000 μ m, for example in high processing property, use the matrix of size between 10-60 μ m, in amboceptor purposes (preparative purpose), use the matrix of size between 50-500 μ m or preferred 50-300 μ m.

Matrix also has a kind of absorbing form, the controlled matrix of density being made up of control density particles coalesce.Above-mentioned coalescent liquefied bed chromatogram in large-scale operation process and non-filling (non-packed) chromatographic column of expanded bed chromatography and various intermittent type chromatograms of being highly suitable for, for example the list in steel basin is criticized absorption process.

In the present invention, affinity ligand can be connected with solid-phase matrix by the known suitable covalent linkage of any kind, the direct chemical reaction by affinity ligand and solid-phase matrix or use known suitable reactant to activate in advance solid-phase matrix or aglucon can be fixed up matrix main component and aglucon.Suitable activating reaction thing has, for example Epicholorohydrin, epibromohydrin, thiazolinyl glycidyl ether (allyl-glycidylether); Di-epoxide is as butanediol diglycidyl ether (butane diol diglycidylether); Halogen divinyl fatty compounds is as dichlorohydrine, divinyl sulfone; N,N'-carbonyldiimidazole; Aldehydic hydrogen is as glutaraldehyde; Quinone; Cyanogen bromide; Periodate is as sodium periodate; Carbodiimide; Chlorotriazine is as cyanuryl chloride; SULPHURYL CHLORIDE is as toluene sulfonyl chloride and Halothane SULPHURYL CHLORIDE (tresyl chlorides); N N-Hydroxysuccinimide; The fluoro-1-picoline of 2-toluene-4-sulfonic acid ester (2-fluoro-1-methylpyridinlum toluene-4-sulfonates); Oxazolone; Maleimide; Two thiopyridines and hydrazides.In these activating reaction things, preferably there is the SP1 of separant group (leaving a spacer group SP1 different from asingle bond) the activating reaction thing that is different from singly-bound, for example Epicholorohydrin, epibromohydrin, propenyl glycidyl ether; Di-epoxide; Halogen divinyl fatty compounds; Divinyl sulfone; Aldehyde; Quinone; Cyanogen bromide; Chlorotriazine; Oxazolone; Maleimide; Two thiopyridines and hydrazides.

The activating reaction thing being concerned by people is especially epoxy compounds, for example Epicholorohydrin, thiazolinyl glycidyl ether and butanediol diglycidyl ether.

In the present invention, all matrix that can fix peptide aglucon can be used in peptide affinity chromatography system.Preferably Fractogel epoxy (M) or " monoblock type chromatographic media " (" monolithic chromatography medium ") CIM-epoxy of Darmstadt, Germany Merck company.Aglucon can directly be fixed in the main component of chemically sensitized chromatography matrix, also can pass through in separant (spacer) or linking agent (linker) and chromatography matrix main component fixing.In the time connecting by separant, the separant being combined on chromatography matrix need pass through chemokinesis, to aglucon is fixed.Preferably Fractogel epoxy matrix is combined with separant.

In a particularly preferred embodiment of the present invention, separant after first reacting with diamino dipropyl amine (diaminodipropylamine (DADPA)) by chromatography matrix again with succinyl oxide (SA) formation that reacts.The terminal carboxyl of the separant forming is through being connected with a terminal is amino after chemokinesis.The reactive group that aglucon is fixed on the separant containing on matrix or by it is fixed on separant.Can be amine groups for the reactive group of peptide aglucon, carboxyl group or sulfydryl group.In the present invention, preferably by ammonia key, peptide is fixed on matrix or separant.

In the present invention, affinity matrix is in particular for the affinity matrix of affinity chromatography material, preferably meets above a) and b) defined oligopeptides.

Term used herein " oligopeptides " means and contains at least three amino acid whose protein compounds.Under normal circumstances, the length of oligopeptides is no more than 35 amino acid lengths, is preferably 4 to 20 amino-acid residues.

The oligopeptides aglucon preferred length that in the present invention, affinity chromatography system is used is 5 to 12 amino acid, more preferably 6 to 8 amino acid, preferably comprise a tryptophan residue, aglucon is optionally fixed on the fusion polypeptide part with self-proteolysis function under chaotropic environment, in the time that environment change is cosmotropic, aglucon still keeps fixing with the part of the fusion polypeptide with self-proteolysis function.

Known to as an example of antibody example, in affinity chromatography, use special the fixing between some polypeptide and other polypeptide.Oligopeptides also can be used as affinity ligand.These molecules have higher chemical stability, effect and selectivity, and have lower price, and are generally avirulent.These characteristics are very large advantages in bio-pharmaceuticals technique.Those skilled in the art will know that and how from combined peptide library or biological library, to find the directly oligopeptides aglucon for target molecule.According to the present invention, the screening of oligopeptides aglucon need to be carried out in chaotropic environment.

In the present invention, significant characteristic of affinity ligand is, it can make under sex change environment, and for example 4M urea, with N ^proand N ^profusion rotein (with and mutation-ure or the protein that comprises its mutation-ure) fix.

The method of the known synthetic peptide of prior art can be used for preparing the oligopeptides aglucon described in the present invention.Preferably by people such as SPOT synthesis methods, PIN synthesis method, teabag synthesis method, Ruiwu Liu the people such as mix andsplit synthesis method or Joseph A.Buettner described in Experimental Hematology 31 (2003) 11-30 at Int.J.Peptide Protein Res.47 (1996), the PELICAN synthesis method described in 70-83 is prepared oligopeptides aglucon.Can use some linking agent chemicals to fix (anchoring) first amino acid.

In a preferred embodiment of the invention, can first prepare respectively aglucon and then be fixed on chromatography matrix, in another preferred embodiment of the present invention, peptide aglucon directly can be synthesized on chromatography matrix.

Oligopeptides aglucon has very strong specificity.In the present invention, the notable feature of the oligopeptides of synthesized is, it can be under the environment that makes sex change and N ^pro, N ^proderivative and fusion polypeptide thereof are connected.In the present invention, oligopeptides is directly for the fusion polypeptide part with self-proteolysis function.

In a preferred embodiment of the present invention, wherein the aminoacid sequence of oligopeptides can be selected from following sequence: VSIFEW, AVSIEWY, AVSFIWY, VSFIWYK, ASRFWYA, AFYTWYA, AFYRWYK, AFYRWY, AFYRWYA, AVSIFEWY, AVSRNWY, ASRFWY, AFYRWYAA, AFYRWY, ASRFWYAA, AFYRWYAA and AFYSWYAA.

In the present invention, oligopeptides aglucon can also can, with a sealing N end, form the N end of sealing with a free N end by for example acetylizing.

In most preferred embodiment of the present invention, use sequence is SEQ ID NO 5 (be called because the derivative of this mutant has at 53 to 57 " EDDIE " partial sequences (substitute " RGDIR " wild-type) this mutant (with other mutation-ures that contain this part) " EDDIE-mutant), the N of natural CSFV ^proderivative combine with the oligopeptides aglucon that is selected from following amino acid sequences: ASRFWYA, AFYTWYA, AFYRWYK, AFYRWY and AFYRWYA.

Preferably affinity ligand is selected from: VSDDWY, VSEDWY, VSIDWY, VSYDWY, VSVDWY, VSWDWY, VSYDWY, VSFDWY, VSDEWY, VSEEWY, VSIEWY, VSYEWY, VSVEWY, VSWEWY, VSYEWY, VSFEWY, DDDDWY, DDEDWY, DDIDWY, DDYDWY, DDVDWY, DDWDWY, DDYDWY, DDFDWY, VSIFWE, FSIFEW, WSIFEW, VSLIWY, VSLIDW, VSLIEW, VSLIWE, FSLEEW, VSDLDW, VSDLEW, VSYIDW, (above-mentioned all amino acid is 5.5 o'clock fix N in pH value to VSYIWE ^pro), VSIDWY, VSIEWY, VSIWWY, VSIIWY, VSYIWY, VSVIWY, VSFIWY, VSFIWE, VSIFEW, VSIFWE, FSIFEW, WSIFEW, VSLIWY, VSLIDW, VSLIEW, VSLIWE, FSLIEW, WSLIEW, FSYFEW, FSFYEW, WSFYEW, FSYIEW, (above-mentioned all amino acid is 7.3 o'clock fix N in pH value to WSYIEW ^pro), AFYTWYA, AFYRWYK, AFYRWY, AFYRWYA, AFFRWYA, AFGRWYA, AFHRWYA, AFIRWYA, AFLRWYA, AFMRWYA, AFNRWYA, AFPRWYA, AFQRWYA, AFRRWYA, AFSRWYA, AFTRWYA, AFVRWYA, AFYRWYA, AFYFWYA, AFYGWYA, AFYLWYA, AFYMWYA, AFYNWYA, AFYPWYA, AFYTWYA, AFYVWYA, AFYWWYA, AFYYWYA, AKWFRYA, VSRNWY, ASRNWYA, ASRFWYA, FSRNWYA, VFRNWYA, VWRNWYA, VYRNWYA, VSRAWYA, VSRFWYA, VSRWWYA, VSRYWYA, VSRNFYA, VSRNYYA, VSRNWFA, (above-mentioned all amino acid is for the N on 53 to 57 amino acids residues with EDDIE sequence for VSRNWWA ^promutant has extremely strong avidity), Ac-AFYTWYAK, Ac-AFYRWYKK, Ac-AFYRWYK, Ac-AFYRWYAK, Ac-AFFRWYAK, Ac-AFGRWYAK, Ac-AFHRWYAK, Ac-AFIRWYAK, Ac-AFLRWYAK, Ac-AFMRWYAK, Ac-AFNRWYAK, Ac-AFPRWYAK, Ac-AFQRWYAK, Ac-AFRRWYAK, Ac-AFSRWYAK, Ac-AFTRWYAK, Ac-AFVRWYAK, Ac-AFYRWYAK, Ac-AFYFWYAK, Ac-AFYGWYAK, Ac-AFYLWYAK, Ac-AFYMWYAK, Ac-AFYNWYAK, Ac-AFYPWYAK, Ac-AFYTWYAK, Ac-AFYVWYAK, Ac-AFYWWYAK, Ac-AFYYWYAK, Ac-AKWFRYAK, Ac-VSRNWYK, Ac-ASRNWYAK, Ac-ASRFWYAK, Ac-FSRNWYAK, Ac-VFRNWYAK, Ac-VWRNWYAK, Ac-VYRNWYAK, Ac-VSRAWYAK, Ac-VSRFWYAK, Ac-VSRWWYAK, Ac-VSRYWYAK, Ac-VSRNFYAK, Ac-VSRNYYAK, Ac-VSRNWFAK, Ac-VSRNWWAK, YWKA, Ac-YWKAK, YKYA, Ac-YKYAK, YWRA, Ac-YWRAK, ARWY, Ac-ARWYK, YWRA, Ac-YWRAK (turns use (lysination) into because N holds acetylizing and C end Methionin, above-mentioned all peptides have improved the ability to cure to substrate).

Any matrix that can fix peptide aglucon can be used in the peptide affinity chromatography in protection domain of the present invention.The preferably Fractogel epoxy (M) of Darmstadt, Germany Merck company or " the monoblock type chromatographic media " of CIM-epoxy.Aglucon can directly be fixed in the main component through chemically sensitized chromatography matrix, also can fix by separant or linking agent.In the time connecting by separant, the separant being combined on chromatography matrix need pass through chemokinesis, to aglucon is connected.Fractogel epoxy matrix is combined with better effects if with separant.

In a preferred embodiment of the invention, first separant (DADPA) is reacted and reacts and get with succinyl oxide (SA) more afterwards with diamino dipropyl amine (diaminodipropylamine) by chromatography matrix.The terminal carboxyl of separant is through being preferably connected with a terminal is amino after chemokinesis.Reactive group aglucon by separant is fixed on matrix, or is fixed on separant.Can be amine groups for the reactive group of peptide aglucon, carboxyl group or sulfydryl group.In the present invention, preferably by ammonia key, peptide is fixed on matrix or separant.

After fusion polypeptide is fixed in chromatogram system, loose impurity component just can wash down from chromatographic column easily.These impurity components may be for example host cell polypeptide and are gathered in inclusion body or are adsorbed on the nucleic acid on inclusion body, after these nucleic acid are dissolved, are still present among polypeptide solution, also have enzyme cytoclasis remaining composition afterwards.After cleaning, be just fixed in chromatographic column only remaining fusion polypeptide, therefore following step is all to carry out in the system of purifying.

Fusion polypeptide is fixed in chromatographic column under environment high from liquid sequence, that make inactivation.After fixing, be cosmotropic environment by environment change, to induce refolding.

In a preferred embodiment, the operation of the refolding of fusion polypeptide is carried out chaotropic environment change to the environment of cosmotropic by changing damping fluid.

Can be little by little or moment chaotropic damping fluid is replaced to the damping fluid of cosmotropic.In a preferred embodiment of the invention, chaotropic damping fluid can be replaced with to cosmotropic damping fluid gradually, the similar stopper of the now effect of damping fluid (plug).In another preferred embodiment of the present invention, also chaotropic damping fluid moment can be replaced with to cosmotropic damping fluid.

If regulate temperature will greatly promote fixing and/or refolding and the shearing of fusion polypeptide in chromatographic column in buffer-exchanged process.Can use the method for for example cooling jacket/heating jacket.Therefore, in a preferred embodiment, use cooling jacket/heating jacket to regulate as temperature; If can in advance the temperature of damping fluid be controlled within required scope better, so just thermoregulator object can be reached.

In the time of environment change in packing layer, fusion polypeptide starts to carry out refolding, and activates it and have the activity of self-proteolysis funtion part.Afterwards, on the position determining, the needed polypeptide of fusion polypeptide C end is sheared in the specificity of self-proteolysis part, therefore required polypeptide has just formed a homology N end.According to the time of fusion polypeptide refolding, in the time that all chaotropic damping fluids are all replaced in packing layer, slowly reduce the speed of mobile phase in cosmotropic damping fluid or stopped.After refolding is carried out, add cosmotropic damping fluid by continuation the needed polypeptide of shearing is cleaned out.The fusion polypeptide wash-out that by traditional method, fusion polypeptide N end is had to the part of self-proteolysis function and do not shear out, for example, uses higher salt concn, changes change pH values or NaOH and makes chromatographic material regeneration.Can clean packing layer with the damping fluid that self-proteolytic enzyme can be peeled off from sorbent material, make chromatographic material regeneration.These damping fluids comprise acid solution, alkaline solution or organic solvent.In packing layer, again add the circulation that initial damping fluid/chaotropic damping fluid just can enter a new round.

Because velocity of shear cannot reach expection height value sometimes, so the fusion polypeptide of not shearing washing down from chromatographic column in the regeneration stage can be back to next circulation again.

According to selected damping fluid difference, the polypeptide of gained may be part refolding or complete refolding.In the present invention, may be part refolding or complete refolding by wash-out required polypeptide out, refolding is best completely certainly.In the present invention, likely there is the part refolding completely of self-proteolysis function in fusion polypeptide, and required not refolding of polypeptide retaining part.When required polypeptide has very complicated structure as dimer or tripolymer, or comprise while making up base (prosthetic group) or cofactor (cofactor), just may produce above-mentioned phenomenon.Need special environment to carry out complete refolding to this kind of required polypeptide.Can be by such as proton power (protonic strength) and pH value or remove in refolding process employings point other steps such as the stain remover that conventionally adds completely and form above-mentioned special environment, make to fold and carried out completely gradually.

In the present invention, can carry out modification arbitrarily to environment makes fusion polypeptide keep being adsorbed in chromatographic column.

The present invention has also set forth for the present invention's oligopeptides aglucon and the N of above-mentioned CSFV ^proderivative.The present invention relates to the N of oligopeptides aglucon and above-mentioned CSFV equally ^propurposes in the present invention of derivative.

Unless stated otherwise, the implication of all technology used and scientific terminology is all identical with implication understood by one of ordinary skill in the art here.

By the following example, the present invention has been carried out further describing, this embodiment only uses with explanation and is unrestricted.Especially, the following example relates to the preferred embodiments of the present invention.

Embodiment

Embodiment 1: by using peptide affinity chromatography and different types of fusion polypeptide to prepare the method for heterologous polypeptide of the present invention.

1.1. use the N of pestivirus ^prooneself's proteolytic enzyme is prepared heterologous polypeptide

The present embodiment has been described pestivirus oneself proteolytic enzyme 6xHis-N ^prothe preparation process of fusion polypeptide GFPmut3.1, thereby, refolding and shear and carry out on peptide affinity matrix.

In following examples, for N ^prothe GFPmut3.1 constructing be the mutant of GPF, GFP is highly suitable for preparing in intestinal bacteria.GFPmut3.1 has following amino acid sequences and substitutes: S2 is replaced by R, and S65 is replaced by G, and S72 is replaced by A.The 178th to 415 amino acids sequences of whole fusion structure are named as 6H-sNp-Gmut3.1-Pet30a, referring to the aminoacid sequence of Gmut3.1.

The structure of 6H-sNp-Gmut3.1-Pet30a carrier has been described in 1.2.1.1 and 1.2.1.2.

The conversion process of host cell has been described in following 1.2.2.

1.1.1 chromatogram arrangement

The chromatogram using in embodiment 1 realizes by AeKTA100Explorer chromatogram system (Amersham Biosciences).The peptide affinity adsorbent preparing is inserted to (5mm i.d., Amersham Biosciences) in HR5 chromatographic column.Gel volume is about 1ml.

1.1.2 the preparation of oligopeptides aglucon

The oligopeptides aglucon using in embodiment 1 is prepared as follows:

The synthetic of solid-phase peptide is at 433A peptide synthesizer (Applied Biosystem, vienna, Austria) in by Fmoc-protected amino acid (Bachem, Bubendorf, Switzerland) 1-hydroxyl-1H-benzotriazole/N, N '-dicyclohexyl carbimide (1-hydroxy-1H-benzotriazol/N, N '-dicyclohexylcarbodiimide (HOBt/DCC)) activation is carried out.Peptide is upper synthetic in 4-methylol-phenoxymethyl-co polystyrene (4-hydroxymethyl-phenoxymethyl-copolystyrene-) 1% divinylbenzene resin (HMP resin, Wang resin).Side chain protected group is the tertiary butyl (T-Bu) of tyrosine, Serine, Threonine; the OtBu of L-glutamic acid, aspartic acid; the tert-butoxy carbonyl (tert-butoxycarbonyl) of Methionin, tryptophane (Boc), and the trityl (Trt) of halfcystine, Histidine, asparagine, glutamine.Use 4-dimethylaminopyridine (DMAP) as catalyzer for first amino acid whose coupling.After first amino acid coupling, use benzoyl oxide to add cap step (capping step).The piperidines of use 20% completes the deprotection of Fmoc base.By containing 95% trifluoroacetic acid (TFA), the deprotection of side chain and the shearing from resin thereof are carried out in the reaction of the sheared mixt of 2.5% water and 2.5% tri isopropyl silane (TIS).After using dichloromethane wash-out, just can purification of crude amino acid (crude peptide) by repeatedly carrying out the aldehyde ether precipitator method and then carrying out lyophilization.Use with P3500 pump (Amersham Biosciences, Uppsala, Switzerland) and Luna15 μ C18 (2) 250 × 21.2mm chromatographic column (Phenomenex, Torrence, Canada, U.S.) RPLC, use acetonitrile and water (0.1%TFA) that linear gradient is 5-50%, its flow is 30ml/min, just can carry out further purifying to amino acid.Use with HP1090 liquid chromatography (Hewlett Pchard, the U.S.) and the analysis RPLC of Luna3 μ C18 (2) 100 × 4.6mm chromatographic columns, use the acetonitrile that linear gradient is per minute 1%, just can determine amino acid whose purity.Laser desorption ionization-time-of-flight mass spectrometer (ThermoBioanalysis, Hempstead, Britain) auxiliary matrix be can pass through and homology and identity measured.

1.1.3 the preparation of affinity matrix

The affinity matrix using in embodiment 1 is prepared as follows:

10g Fractogel epoxy (M) (Merk, Darmstadt, Germany) reacts 48 hours at ambient temperature with the diamino dipropyl amine (Diaminodipropylamine (DADPA)) of 50ml 1M.React complete after, clean gel with the HCL of 50ml 10mM and the water of 3 times 50ml.Gel is resuspension in water, adds the NaOH of 0.1M and 2g succinyl oxide (succinic anhydrid) that pH value is adjusted to 7.0.After mild stirring 30min, add again the NaOH of 10M and 2g succinyl oxide (succinic anhydrid) to make pH value reach 7.0.After passing through again the mild stirring of 30min, use the NaOH of 50ml 0.1M, the phosphate buffered saline (PBS) of 50ml, the water of three times 50ml and 20% ethanol clean gel.Under 4 ℃ of conditions, preserve gel afterwards through suction dried (suction drying).

1.1.4 the activation of carboxyl and peptide is fixing

Affinity matrix in embodiment 1 is activated by the following method:

Described in 1.1.3, after the wet Fractogel of 1g modifies through DADPA-SA separant (spacer), then with twice of the acetonitrile cleaning of 5ml.Activation by dissolving the triethylamine of the succinimido trichloroethane carbonic ether of 3ml 0.1M (Succinimidyl trichloroethyl carbonate) and 0.1M and carried out for 3 hours in acetonitrile.Finally use the HCL of acetonitrile and 1mM to clean gel.Peptide AFYRWYA is dissolved in PBS with the concentration of 3mg/ml.Rapidly 5ml peptide solution added in gel and make its reaction 24 hours.Peptide VSFIWYK is dissolved in the dimethyl formamide (DMF) that contains 0.1M triethylamine.5ml peptide solution is added rapidly in gel and make its reaction 24 hours.By just determining coupling productive rate to carrying out rp-hplc analysis before and after sample coupling.

Peptide fixing on CIM-epoxy:

The pH value that peptide is dissolved in to 100mM is 10.0, the Na of the NaCL that contains 0.15M ₂cO ₃in damping fluid.CIM-dish is provided in the chuck (cartrige) being provided by manufacturer, at room temperature uses P1 pump (Amersham Biosciences) circulate slowly to extract peptide solution to make it through CIM-dish lasting 48 hours of this process.By just determining coupling productive rate to carrying out rp-hplc analysis before and after sample coupling.The block (block) that uses the thanomin of 0.5M remaining epoxy group(ing) to be carried out 48 hours under the condition that coupling is 10.0 in pH value after completing.

1.1.5 the expression of fusion polypeptide

The N that contains the GFPmut3.1 with 6xHis-tag and the fusion of C end the fermentor cultivation of 10 liters ^prothe self-proteolytic enzyme of N end, expresses fusion polypeptide intestinal bacteria recombinant chou HMS174 (DE3).The aminoacid sequence of fusion polypeptide is as follows:

SEQ ID NO 23：

1 MHHHHHHELN HFELLYKTSK QKPVGVEEPV YDTAGRPLFG NPSEVHPQST LKLPHDRGRG 60

61 DIRTTLRDLP RKGDCRSGNH LGPVSGIYIK PGPVYYQDYT GPVYHRAPLE FFDEAQFCEV 120

121 TKRIGRVTGS DGKLYHIYVC VDGCILLKLA KRGTPRTLKW IRNFTNCPLW VTSCSGTMRK 180

181 GEELFTGVVP ILVELDGDVN GHKFSVGGEG EGDATYGKLT LKFICTTGKL PVPWPTLVTT 240

241 FGYGVQCFAR YPDHMKQHDF FKSAMPEGYV QERTIFFKDD GNYKTRAEVK FEGDTLVNRI 300

301 ELKGIDFKED GNILGHKLEY NYNSHNVYIM ADKQKNGIKV NFKIRHNIED GSVQLADHYQ 360

361 QNTPIGDGPV LLPDNHYLST QSALSKDPNE KRDHMVLLEF VTAAGITHGM DELYK

Bacterial host cell, namely expression strain (expression strain), its cultivation is to put into practice (microbiological practice) according to known micro-biological process to carry out.Generally, expression strain is cultivated and is formed on substratum by mono-clonal, can certainly use the cell suspending liquid (cell bank) of low temperature.Conventionally need to cultivate bacterial strain through multi-stage process and obtain enough biomasss for future use.

In small-scale cultivation situation, can in shaking flask, carry out, most applications is used complex medium (for example LB broth culture) to cultivate.Also can use the culture medium culturing base (for example citrate medium) of definite ingredients.First to carry out to host cell (cell suspending liquid that uses mono-clonal inoculation or low temperature to cultivate (cryo-culture) is inoculated) preculture (pre-culture) of small volume, generally, temperature in this culturing process is not crucial for follow-up expression of results, thus usual at relatively high temperature (for example 30 ℃ or 37 ℃) cultivate.Main culturing process is for example, to carry out in larger volume (500ml), and this process especially needs to guarantee good ventilation (with the volume contrast of inclusion, high speed rotating large volume shaking flask).Because hope expression is carried out with the form of insoluble inclusion body, for example, so in most of situation, main culturing process also need be carried out at relatively high temperature (30 ℃ or 37 ℃).Inducible system is highly suitable for preparing inclusion body (for example containing trp, lac, tac, phoA promotor).In the time reaching logarithmic growth late period (late logarithmic phase) (being generally in the time that the optical density (OD) in shaking flask is 0.5 to 1.0), add hatch (incubation) that inductor (for example indoles acrylic acid, isopropyl-β-D-thiogalactoside(IPTG)=IPTG) then continues to carry out 1 to 5 hour.During this period, most N ^profusion polypeptide precipitates in tenuigenin with the form of inclusion body.Can collect the cell of final formation and it is further processed.

In large scale culturing situation, multilevel hierarchy is made up of a large amount of bio-reactor (fermentor tank), preferentially uses in this case the substratum of definite ingredients to improve the process engineering control (process engineering control) of whole method.In addition, can also greatly increase biomass and turnout by quantitatively add special nutrition ((fed batch) in batches) in substratum.In addition, this technique is similar with use shake-flask culture.For example, use a primary fermentation tank and a main fermentor tank, select and culture temperature similar in flask process.In fermentor tank, use is cultivated by mono-clonal or low temperature culture (cryoculture) the so-called inoculum (inoculum) getting and is inoculated (inoculate) in shaking flask in the early stage.In fermentor tank, especially in main culturing process, must also guarantee good ventilation and sufficient inductor concentration.But in some cases, the time of inductive phase must obviously be greater than the time in shaking flask process.The final cell forming is further processed.

1.1.6 the separation of inclusion body

After collecting the final cell forming, cell (weight in wet base 850g) is suspended in to the Tris/HCL that pH value is 8.0 the 50mM that contains 2500ml, in the solution of the EDTA of 5mM and 1% TritonX-100.Cold suspension is broken cell by APV-2000 high pressure homogenisers (Invensys) three times under the pressure of 800bar.Through in the process of high pressure homogenisers, with ice cube frozen suspension liquid, make cell homogenization with Ultraturrax.Homogenate is carried out to low speed (JLA10.500,7500rpm, 30min) centrifugation to obtain the inclusion body that contains recombinant fusion polypeptide.

1.1.7 the dissolving of inclusion body

Rounded grain (pellet) is suspended in to the Tris/HCL that pH value is 8.0 the 50mM that contains 2500ml, in the solution of the EDTA of 5mM and 1% Triton X-100, then carries out centrifugation.Repeat this step.After water-washing step, rounded grain (pellet) is just suspended in water.The inclusion body suspensions obtaining is stored under the environment of-20 ℃ in order to further using.At ambient temperature, use the solution of the DTT that contains 50mM Tris/HCL, 10M urea and 50mM that pH value is 7.3 with the ratio of 1: 5, inclusion body suspensions to be diluted.By centrifugation, undissolved composition is cleared out.Acquisition is about the peptide concentration of 15mg/ml.The solution of the NaCL that contains 50mM Tris/HCL, 100mM that use pH value is 7.3 and the urea of 4M dilutes polypeptide solution until peptide concentration reaches about 2mg/ml.

1.1.8 fusion polypeptide fixing in chromatographic column

The polypeptide solution of 0.5ml is added in Fractogel-DADPA-SA-VSFIWYK (0.5 × 0.5cm) matrix, and the preparation of this polypeptide and combination are carried out according to the method described in 1.1.2 and 1.1.3 whereby.Use the NaCl of 50mM Tris/HCL, 100mM and the urea of 4M that pH value is 7.3, with the flow velocity of 50cm/h, chromatographic column is carried out to balance.After injecting sample, this flow velocity is increased to 150cm/h.

1.1.9 do not fix the cleaning of impurity

Use the level pad of 5 times of chromatographic column volumes to clean frozen composition not.Be replaced by afterwards refolding damping fluid, the damping fluid of the DTT of the EDTA of the Tris/HCl that contains 0.5M, 2mM that particularly pH value is 7.3,3% glycerol and 5mM, this damping fluid volume is 4.5 times of chromatographic column volumes.

1.1.10 refolding, shearing and wash-out

By chaotropic environment change to cosmotropic environment, by stopping the mobile refolding that fusion polypeptide is carried out 25 hours on chromatography resin of damping fluid.Active self-proteolytic enzyme is sheared the GPFmut3.1 of C end get off.Use refolding damping fluid to carry out wash-out with the low flow velocity of 50cm/h, determine through fluorometric investigation device and SDS-PAGE, the material eluting is pure natural GPFmut3.1.

1.1.11 regeneration

The NaOH that uses 0.1M, carries out regenerative operation with the low flow velocity of 50cm/h to chromatography resin.

1.2 use the N of pestivirus ^prothe derivative of oneself's proteolytic enzyme is prepared heterologous polypeptide

The present embodiment has been described pestivirus N ^promutant (the 6xHis-N of oneself's proteolytic enzyme ^proeDDIE) preparation of fusion polypeptide GPFmut3.1, in this preparation process, refolding and enzyme are cut all and are carried out on peptide affinity matrix.

The preparation of oligopeptides aglucon and affinity matrix, described in embodiment 1.1, is used chromatogram arrangement in the same manner as in Example 1.

1.2.1 the structure of plasmid

1.2.1.17H-Np-Gmut3.1-pET30a the structure of plasmid

One contains (N-terminally truncated), the N end of clipping N end with the N of 7-His tag ^prothe DNA fragmentation of gene is from the enterprising performing PCR amplification of NP6-pET (Sandoz) plasmid, and be inserted into pET-30a (#69909-3 via Ndel and Kpnl (Asp718) restriction site (restriction sites), 2002-2003 catalogue, Novagen, CN Bioscience company, Merck KgaA, Darmstadt, Germany) in.

Primer pair:

T7-pET(SEQ ID NO 24)：

5′-GAA ATT AAT ACG ACT CAC TAT AGG -3′；

N ^pro R-Kpn(SEQ ID NO 25)：

5 '-ATA CGG TAC CAG AGC AAC TAG TTA CCC ATA ATG-3 ' reacts (ligation reaction) by joint and transforms escherichia coli DH5a lpha (#10643-013, Invitrogen catalogue 2003, Invitrogen Life Technologies Corporation, 1600Faraday Avenue, PO Box 6482 Carlsbad, California, 92008), from the clone body transforming, separate DNA plasmid, confirm that through order-checking gained is 7H-N ^pro-pET30a plasmid.GFPmut3.1 plasmid also can pass through pGFPmut3.1 plasmid (#6039-1, catalogue1999, BD Biosciences Clonetech, 1020 East Meadow Circle, Palo Alto, Canadian 94303-4230, the U.S.) carry out pcr amplification.

Primer pair:

GFP F-Kpn(SEQ ID NO 26)：

5′-GAA AGG TAC CAT GCG TAA AGG AGA AG-3′

GFP R-Sal(SEQ ID NO 27)：

5 '-TAA GTC GAC TTA TTT GTA TAG TTC ATC CAT GGC-3 ' emanates by gel and is cloned into 7H-N via Kpnl-Sa/l restriction site (restriction sites) ^proin-pET30a structure, result forms SGT (Serine-Padil-Threonine) aminoacid sequence after shearing site.Verified, the aminoacid sequence of 7H-Np-Gmut3.1-pET30a structure is as implied above.

1.2.1.2 the structure of 6H-sNp-Gmut3.1-pET30a plasmid

N ^pro-insulinogenic DNA sequence dna (SEQ ID NO 28):

ATGGAACTCAATCATTTCGAACTGCTCTACAAAACTAGCAAGCAAAAACCTGTTGGCGT

TGAAGAGCCGGTCTACGATACTGCAGGTCGTCCTCTTTTTGGGAATCCGTCCGAAGTG

CACCCCCAGTCAACCCTCAAGCTTCCCCATGACCGCGGACGCGGTGACATTCGTACAA

CGCTGCGCGATCTGCCTCGTAAAGGCGATTGTCGCTCTGGAAACCACCTAGGTCCGGT

GTCGGGCATTTACATTAAACCAGGTCCCGTCTATTACCAAGACTACACTGGTCCGGTTT

ACCATCGTGCACCTCTGGAATTCTTTGATGAAGCTCAATTTTGCGAAGTGACTAAACGT

ATTGGCCGTGTAACCGGTTTCGGACGGGAAACTGTACCACATCTACGTGTGCGTTGATG

GCTGTATCCTGCTGAAACTCGCGAAGCGCGGAACCCCTCGCACCCTGAAATGGATCCG

TAACTTCACTAACTGTCCACTGTGGGTCACTAGTTGCTTCGTTAACCAACATCTGTGCG

GTTCACACCTTGTGGAAGCCCTGTATCTGGTGTGTGGCGAACGCGGATTCTTTTATACC

CCGAAAACGCGGCGCGAAGCCGAAGATCTTCAGGTTGGTCAAGTGGAACTGGGCGGA

GGTCCGGGAGCCGGGAGGGTGCAACCGCTGGCGCTTGAAGGGTCGCTGCAAAAACGC

GGTATTGTTGAACAGTGCTGTACCTCCATCTGCTCTCTGTATCAGCTGGAAAACTACTG

CAATTAATAA

This DNA sequence dna is through customization synthetic (custom-synthesized) and be inserted into (the 1000 Atlantic Avenue of Operon Biotechnologies company, Suite 108 Alameda, Canada 94501, the U.S.) pUC119 (NCBI#U07650:National Center forBiotechnologY Information Plasmid Database, National Library ofMedicine, Building 38A, Bethesda, Maryland 20894, the U.S.) in.From this plasmid, the N that runic represents ^prothe sequence of-proinsulin (pro-insulin) can be carried out pcr amplification by following primer pair:

6H-N ^pro-F-Ndel(SEQ ID NO 29)：

5′-CTC TCA TAT GCA TCA CGA TCA TCA TGA CGA ACT CAA TCA TTT CGA ACT GCT C-3′

And Ins-R-Sall (SEQ ID NO 30):

5′-CTT TCG TCG ACT TAT TAA TTG CAG TAG TTT TC-3′

Separate the final fragment obtaining with gel extraction (gel extraction) by agarose gel electrophoresis, and join pET30a carrier (#69909-3 to by the new Ndel forming and the restriction site (restrictionsites) of Sall (bold-type letter), 2002-2003 catalog, Novagen, CN Biosciences company, Merk KgaA, Darmstadt, Germany) in, and shear and form 6H-sN at same restriction site place ^pro-Ins-Pet30a.Described 6H-sN ^pro-Ins-Pet30a is sheared on the restriction site of Spel and Sa/l, by gel electrophoresis and gel extraction (extraction), longer fragment is separated, thereby is deleted proinsulin sequence.Preparation work before inserting is to use identical enzymic digestion carrier 7HNp-Gmut3.1-pET30a (structure is shown in 1.2.1.1) and use gel extraction (gel extraction) that the DNA fragmentation accurately of coding GFPmut3.1 is separated.DNA fragmentation is bonded in ready carrier, obtains 6H-sNp-Gmut3.1-pET30a structure.Its DNA sequence dna is as shown in 1.2.1.1.

1.2.1.3 the structure of S-Np-Ins-Pet30a

To containing N ^prothe structure of the DNA sequence dna of-pro-insulin customizes synthetic (custom-synthesized), and is inserted in the pUC119 of Operon Biotechnologies company.Use following primer pair to required N ^prothe sequence of-pro-insulin is carried out pcr amplification:

N ^pro-F-Ndel(SEQ ID NO 31)：

5′-CGCGACATATGGAACTCAATCATTTCGAAC-3′

And Ins-R-Sall (SEQ ID NO 30)

Separate the final fragment forming with gel extraction through agarose gel electrophoresis, and be bonded in carrier pET30a by the new Ndel forming and the restriction site of Sall (bold-type letter), and shear at identical restriction site.SN ^prothe DNA sequence dna of-Ins-pET30a plasmid is as shown in 1.2.1.1.

1.2.1.4 the structure of 6H-EDDIE-sGmut3.1-Pet30a plasmid

To containing N ^prothe structure of the DNA sequence dna of-pro-insulin customizes synthetic, and is inserted in the pUC119 of Operon Biotechnologies company.Use following primer pair to required N ^prothe sequence of-pro-insulin is carried out pcr amplification:

N ^pro-F-Ndel(SEQ ID NO 31)

With

N ^pro-R-Sall(SEQ ID NO 32)：

5 '-CGC AGA GAT GTT GGT CGA CGC TGC AAC TAG TG-3 ' is inserted into form in pET30a carrier via the Ndel of new formation and the restriction site of Sall (bold-type letter) and forms S-NP-6H-pET30a.S-Np-6H-pET30a is used to two standards, 50 μ l PCR reaction amplification N ^prosequence: first reaction is used the N of 50pmol ^pro-F-Ndel primer (in table 1) and 50pmol return to mutant primer (3 '-), Taq archaeal dna polymerase (#GC002004,2004catalog, the Genecraft of 5 units, Treskow street 10, D-48163 munster, Germany), 1 × PCR damping fluid (#GC 00206,2004 catalog, and the various dNTP mixtures of 20nmol (#GC 013004,2004 catalog, Genecraft) Genecraft); Second reaction used the N of 50pmol ^pro-R-S primer sees the following form 1) and 50pmol forward mutation primer (5 '-), the Taq archaeal dna polymerase of 5 units, the various dNTP mixtures of 1 × PCR damping fluid and 20nmol.PCR reaction is carried out in the thermal cycling cover of a heating, uses follow procedure: 94 ℃: 3min in it; 25 circulations: 94 ℃: 30sec, 54 ℃; 30sec, 68 ℃: 1min; Finally at 68 ℃, hatch 7min.According to the suggestion (QIAquick of manufacturer

spin handbook, in July, 2002) use QIAquick PCR Purification Kit (QIAGEN

limited-liability company, Qiagen street 1, D40724 Hilden, Germany, Cat.Nr.28106, Qiagen Product Guide 2004) the free primer of removing.Centesimal two kinds of PCR are joined together and use the N of 50pmol ^prothe N of-F-Ndel primer (SEQ ID NO.31) and 50pmol ^protaq archaeal dna polymerase (Genecraft), 1 × PCR damping fluid (Genecraft) and the various dNTP mixtures of 20nmol (Genecraff) of-R-Sall primer (SEQ ID NO 32), 5 units, by amplification in its cover of thermal cycling in heating, use follow procedure: 94 ℃: 3min in thermal cycling cover; 25 circulations: 94 ℃: 30sec, 54 ℃: 30sec, 68 ℃: 1min; Finally at 68 ℃, hatch 7min.Use QIAquick PCR PurificationKit (QIAGEN) to remove free primer according to manufacturer's suggestion.Via Ndel and Sall restriction site, PCR fragment is inserted in pET30a carrier.This structure is used to the step of suddenling change afterwards.Amino acid is by the change one by one of six coherent steps, and the selection of primer as shown in Table 1 below separately.Can control in each step plasmid out by above-mentioned DNA sequence analysis (seeing 1.2.1.1).React last sudden change step (I155T and F158T) by the simple PCR of following primer pair.

N ^pro-F-Ndel(SEQ ID NO 31)

With

3′_I155T，F158T(SEQ ID NO 33)：

5′-GCA ACT AGT GAC CCA CAG TGG ACA GTT AGT GGT GTT ACG GGT CCA TTTCAG G-3′

The PCR product obtaining is inserted in S-Np-6H-pET30a via Ndel and Spel (runic sees the following form 1) restriction site.Can determine the sequence of EDDIE-6H-pET30a structure by above-mentioned DNA sequence analysis (1.2.1.1).

Table 1:

5′_C112E	SEQ ID NO 34：GCT CAA TTT GAG GAA GTG ACT AAA CG
		3′_C112E	SEQ ID NO 35：CGT TTA GTC ACT TCC TCA AAT TGA GC
5′_C134E	SEQ ID NO 36：CAT CTA CGT GGA GGT TGA TGG C
		3′_C134E	SEQ ID NO 37：GCC ATC AAC CTC CAC GTA GAT G
5′_C138E	SEQ ID NO 38：GTT GAT GGC GAG ATC CTG CTG
		3′_C138E	SEQ ID NO 39：CAG GAG GAT CTC GCC ATC AAC
5′_A109T，V114T	SEQ ID NO 40：CTG GAA TTC TTT GAT GAA ACC CAA TTT GAG GAA ACC ACT AAA CGT ATT GG
		3′_A109T，V114T	SEQ ID NO 41：CCA ATA CGT TTA GTG GTT TCC TCA AAT TGG GTT TCA TCA AAG AAT TCC AG
5′_R53E，G54D， R57E	SEQ ID NO 42：CAT GAC CGC GGA GAA GAT GAC ATT GAA ACA ACG CTG C
		3′_R53E，G54D， R57E	SEQ ID NO 43：GCA GCG TTG TTT CTT TGT TCAC CTT CTC CGC GGT CAT G
5′_L143Q	SEQ ID NO 44：GAT CCT GCT GAA ACA GGC GAA GCG CGG AAC
		3′_L143Q	SEQ ID NO 45：GTT CCG CGC TTC GCC TGT TTC AGC AGG ATC

On EDDIE-6H-pET30a, use following primer pair to carry out PCR expansion to it:

Can use 6H-N ^pro-F-Ndel, (SEQ ID No 29) and N ^pro-R-Sal, (SEQID NO 32) and the final fragment forming, replace N via the constructional Ndel of S-Np-Ins-pET30a (seeing 1.2.1.3) and Spel (bold-type letter) restriction site ^pro, form 6H-EDDIE-Ins-pET30a.6H-EDDIE-Ins carrier is digested by Spel/Sa/l, goes out longer fragment by gel electrophoresis and gel extraction, thereby deletes proinsulin sequence.Insertion process is in the pUC119 of Operon Biotechnologies company customization preparation, to synthesize the textural of sGFPmut3.1 gene (SEQ ID NO 46) from containing, using following primer pair (SEQ IDNO 47 and SEQ ID NO 48) to be achieved by PCR.

SEQ ID NO 46：

TGCAGCAAAGGCGAAGAACTGTTTACCGGTGTGGTGCCGATTCTGGTGGAACTGGATG

GCGATGTGAACGGTCATAAATTTAGCGTGAGCGGCGAAGGTGAAGGCGATGCGACCTA

TGGTAAACTGACCCTGAAATTTATTTGCACCACCGGCAAACTGCCGGTGCCGTGGCCG

ACCCTGGTGACCACCTTTGGTTATGGCGTGCAGTGCTTTGCGCGCTATCCGGATCACA

TGAAACAGCATGATTTTTTTAAAAGCGCGATGCCGGAAGGTTATGTGCAGGAACGCACC

ATTTTTTTTAAGATGATGGCAACTATAAAACCCGCGCGGAAGTGAAATTTGAAGGTGAT

ACCCTGGTGAACCGCATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGTAACATTCT

GGGCCATAAACTGGAATATAACTATAACAGCCATAACGTGTATATTATGGCGGATAAAC

AGAAAAACGGTATTAAAGTGAACTTTAAAATTCGCCATAACATTGAAGATGGCAGCGTG

CAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGTGATGGCCCGGTGCTGCTGC

CGGATAACCATTATCTGAGCACCCAGAGCGCGCTGAGCAAAGATCCGAACGAAAAACG

CGATCACATGGTGCTGCTGGAATTTGTGACCGCGGCGGGTATTACGCATGGCATGGAT

GAACTGTATAAATAATAA

sGFP-F-Spe，(SEQ ID NO 47)：

5′-GGA TCC ACT AGT TGC AGC AAA GGC GAA G-3′

With

sGFP-R-Sal(SEQ ID NO 48)：

5′-CGA GGT CGA CTT ATT ATT TAT ACA GTT CAT C-3′.

Afterwards, the PCR product of purifying is digested by Spel/Sa/l and is engaged (ligated) in the Spel/Sa/l of 6H-EDDIE-Ins fragment, uses sGmut3.1 to substitute proinsulin base, because constructing to form 6H-EDDIE-sGmut3.1-pET30a.DNA sequence dna in each step as mentioned above (seeing 1.2.1.1) is controlled.

1.2.2 transform

On the bacteria culture medium of 1 liter, cultivate Electroporation-competent cells and (under 37 ℃, 225rpm condition, grow to OD ₆₀₀=0.5).With ice cube frozen cell suspension 15 minutes (continuously stirring), precipitation (pelleted) (4 ℃, 2500g, 10min) Remove All supernatant liquor afterwards.Under 4 ℃ of conditions, remaining rounded grain (pellet) is suspended in again in the deionized water of 1 liter, slow down (4 ℃ of stirring velocitys (spun down), 2500g, 10min) and by (4 ℃ of step with centrifugal separation intermittently, 2500g, 10min) use the deionized water (4 ℃) of 50ml to clean twice.Finally use 10% the glycerol solution of having sterilized (4 ℃) of 50ml to clean rounded grain, precipitate (4 ℃, 2500g, 10min) afterwards it is suspended in again in 10% the glycerol solution of having sterilized of 2.5ml (4 ℃), then it is freezing and be stored under-80 ℃ of conditions with the equal portions of 40 μ l.Use ice cube that the Electroporation-competent cells of one equal portions is thawed, carry out again the joint reaction of the DNA that includes 5ng of 1 μ l, and send it in the electroporation test tube that interelectrode distance is 1mm (electroporation cuvette) not having in alveolate situation.Use includes BIO-RAD pulse manipulator (Bio-Rad Laboratories company, 2000 Alfred NobelDrive, Hercules, Canada 94547, the U.S.; Cat.n.1652077, Life ScienceResearch Products 1998) BIO-RAD gene pulse producer (Bio-RadLaboratories company, 2000 Alfred Nobel Drive, Hercules, Canada 94547, the U.S.; Cat.n.1652098, Life Science Research Products 1998) carry out electroporation, its intrinsic parameter is set to 1.5kV, 25 μ F, 200Ohms, time constant is no more than 4.5ms.After this add immediately the TY substratum (peptone of 1.0%w/v of 180 μ l, the yeast extract of 0.7%w/v, the NaCl of 0.25%w/v), suspension moved in the plastics tubing of having sterilized of 14ml and hatch 30min (37 ℃, 225rpm).Afterwards suspension is placed on and selects on substratum.Just can obtain clone through 37 ℃ of hatching of spending the night, be moved to the TY substratum of 2ml and overnight incubation under 37 ℃, the condition of 225rpm.Use the overnight culture of 1ml to prepare plasmid by standard method, plasmid prepare the domination of conditionality analysis (restriction analysis) and DNA sequencing.Be used to further transform (transformation) in expression strain through plasmid after sequential analysis, its method will be set forth in this article.

1.2.3 the expression of fusion polypeptide

Recombination bacillus coli HMS 174 (DE3) comprises a pET30 plasmid of expressing fusion polypeptide, and this fusion polypeptide has N and holds self-proteolytic enzyme 6H-N ^proeDDIE, C end is GFPmut3.1, and its aminoacid sequence is as follows, and the cultivation of above-mentioned recombination bacillus coli is to carry out in the shaking flask of LB substratum that is equipped with 1.8 liters.

SEQ ID NO 49：

1 MHHHHHHELN HFELLYKTSK QKPVGVEEPV YDTAGRPLFG NPSEVHPQST LKLPHDRGED 60

61 DIETTLRDLP RKGDCRSGNH LGPVSGIYIK PGPVYYQDYT GPVYHRAPLE FFDETQFEET 120

121 TKRIGRVTGS DGKLYHIYVE VDGEILLKQA KRGTPRTLKW TRNTTNCPLW VTSCSKGEEL 180

181 FTGVVPILVE LDGDVNGHKF SVSGEGEGDA TYGKLTLKFI CTTGRLPVPW PTLVTTFGYG 240

241 VQCFARYPDH MKQHDFFKSA MPEGYVQERT IFFKDDGNYK TRAEVKFEGD TLVNRIELKG 300

301 IDFKEDGNIL GHKLEYNYNS HNVYIMADKQ KNGIKVNFKI RHNIEDGSVQ LADHYQQNTP 360

361 IGDGPVLLPD NHYLSTQSAL SKDPNEKRDH MVLLEFVTAA GITHGMDELY K

The cultivation of cell is described in 1.1.5.

1.2.4 the separation of inclusion body

The fragmentation of cell is achieved by enzyme.In brief, the EDTA of the Tris/HCl that contains 20mM, the 5mM that is 8.2 by cell suspension in the pH of 40ml value, the MgCl of 2mM ₂solution in.Add the lytic enzyme of 72mg and the Benzonase of 300U .After at room temperature hatching 45min, add 1.3gNaCl and 0.5ml Triton X-100.After 15min, suspension is carried out to centrifugation (Beckman JA25.50,10000rpm, 15min, 4 ℃) to obtain inclusion body.

1.2.5 the dissolving of inclusion body

Use 0.5% the Septochol of 20ml once to clean rounded grain, the NaCl that re-uses the 1M of 20ml carries out twice cleaning to rounded grain, and water cleans again afterwards.Remaining rounded grain (weight in wet base is about 2g) is suspended in the water of 10ml and is kept under-20 ℃ of conditions and does further to use.

The solution of the urea of the Tris/HCl that contains 50mM, 10M that use pH value is 7.3 is with the suspension of dilution proportion one equal portions of 1: 5, thus dissolving inclusion body.Centrifugation is removed and re-used pH value after insoluble composition is that the solution of 7.3 the NaCl of the Tris/HCl that contains 50mM, 100mM and the urea of 4M was with the above-mentioned solution of dilution proportion of 1: 5.

1.2.6 inclusion body is fixing

The solution that the content of peptides of 2ml is about to 2mg/ml adds on peptide affinity matrix by aforesaid method.In brief, use the solution equilibria Fractogel-DADPA-SA-AFYRWYA of the urea of NaCl, the 4M of the Tris/HCl that contains 50mM, 100mM that pH value is 7.3.Inject the sample of 2ml with the low speed of 25cm/h.

1.2.7 the cleaning of loose impurity

Use the level pad of 5 times of chromatographic column volumes to clean frozen composition not, flow velocity is 150cm/h.Be replaced by afterwards pH value and be the EDTA of 7.3 the Tris/HCl that contains 200mM, 2mM, the damping fluid of 10% glycerol carries out refolding process, this damping fluid volume is

4.5 times of chromatographic column volumes.

1.2.8 refolding, shearing and wash-out

Stop flowing of damping fluid, make fixing fusion polypeptide carry out the refolding of 25h.In refolding process, fusion composition (fusion partner) GFPmut3.1 is sheared and discharged to fixing proteolytic enzyme at its specific site.Using afterwards pH value is that the EDTA of 7.3 the Tris/HCl that contains 200mM, 2mM, the damping fluid of 10% glycerol carry out wash-out with the flow velocity of 150cm/h to fusion polypeptide.Collect the fragment of 1ml and under 280nm light absorption ratio, 488nm fluorescence and 520nm light emission condition, it analyzed.The fragment that still contains fusion composition GFPmut3.1 carries out further analyzing the GFPmut3.1 to obtain purifying by SDS-PAGE.

1.2.9 regeneration

After eluting the required polypeptide of shearing, need to use the NaOH solution of the 0.1M of 5 times of chromatographic column volumes, with the flow velocity of 150cm/h, chromatographic column is carried out to regenerative operation.

Embodiment 2:

Adopt fixing metal example affinity chromatography to prepare the method for heterologous polypeptide

The present embodiment has been described pestivirus N ^prooneself's proteolytic enzyme (6xHis-N ^proeDDIE) preparation of the fusion polypeptide GFPmut3.1 of mutant, in this preparation process, refolding and shearing are all carried out on fixing metal ions chromatography matrix.

His mark is introduced in fusion polypeptide and makes can use identical structure in fixing metal ions affinity chromatography and polypeptide affinity chromatography, to two kinds of methods are directly contrasted.In affinity chromatography, the interaction of fusion polypeptide and few tire aglucon does not need mark.

The preparation of inclusion body and dissolving are described in embodiment 1.2.4 and 1.2.5.

The preparation of 2.1 chromatographic columns, fusion polypeptide fixing in chromatographic column

Chelating Sepharose Fast flow (Amersham Biosciences) is inserted in chromatographic column with layer (bed) size of 0.5i.d. × 5cm, and water cleans out storage solution.Then by metal ion Ni ²⁺be loaded in chromatographic column.Use the NiCl of the 100mM of approximately 2/3rds chromatographic column volume ₂or NiSO ₄.Loose Ni ²⁺ion is cleaned out.After using the solution equilibria chromatographic column of urea of NaCl, the 4M of the Tris that contains 50mM, 100mM that pH value is 7.3, the polypeptide solution that 0.5ml content is about 2mg/ml can be fixed in chromatographic column.Loading flow velocity (loading flow rate) is 50cm/h.

The cleaning of 2.2 loose impurity

Use the level pad of 5 times of chromatographic column volumes will be after frozen composition does not clean out, it be 7.3 the Tris/Acetate that contains 500mM that level pad is changed into pH value, the sucrose of 0.25M, the damping fluid of the DTT of 1mM.

2.3 refoldings, shearing and wash-out

After using the damping fluid of 4.5 times of chromatographic column volumes, stop flowing of damping fluid, make fusion polypeptide carry out refolding, in refolding process, self-proteolytic enzyme will merge composition (fusion partner) and shear.The flow velocity that reuses 1ml is 150cm/h, the damping fluid of the sucrose of Tris/Acetate, 0.25M that what pH value was 7.3 contain 50mM, the DTT of 1mM just separable go out required polypeptide.Hoarding segment, and use fluorescence measuring device and SDS-PAGE to analyze it.

2.4 regeneration

Use flow velocity is 50cm/h, and the chlorination guanidine that what pH value was 3.5 contain 50mM acetate, 6M is regenerated to chromatography resin.

Embodiment 3:

N ^prothe shearing of EDDIE-sSPA-D in chromatographic column

The present embodiment has been described by pestivirus N ^prooneself's proteolytic enzyme (N ^prothe expressing fusion protein (expression) of mutant EDDIE) is prepared the process of staphylococcal protein A,SPA matter D structural domain, and refolding and shearing are all carried out on polypeptide affinity matrix.Comprise N by following method preparation ^proeDDIE oneself's proteolytic enzyme and C end are connected with the N that is referred to as of A 3-protein d structural domain (sSPA-D) ^prothe fusion rotein of EDDIE-sSPA-D.

Recombination bacillus coli HMS174 (DE3) comprises a pET30 plasmid of expressing fusion polypeptide, and its aminoacid sequence is as follows, and described in 1.1.5, this recombinant chou is cultivated in 101 fermentor tank.1 to 168 amino acids of whole fusion constructs is N ^prothe sequence of EDDIE, the sequence that 169 to 229 amino acids are sSPA-D.

1 11 21 31 41 51

| | | | | |

1 MELNHFELLY KTSKQKPVGV EEPVYDTAGR PLFGNPSEVH PQSTLKLPHD RGEDDIETTL 60

61 RDLPRKGDCR SGNHLGPVSG IYIKPGPVYY QDYTGPVYHR APLEFFDETQ FEETTKRIGR 120

121 VTGSDGKLYH IYVEVDGEIL LKQAKRGTPR TLKWTRNTTN CPLWVTSCAD AQQNKNNKDQ 180

181 QSAFYEILNM PNLNEEQRNG FIQSLKDDPS QSTNVLGEAK KLNESQAPK

The separation of inclusion body and dissolving are described in embodiment 1.1.Fusion polypeptide fixing in chromatographic column

Use flow velocity is 150crn/h, pH value is solution equilibria Fractogel-DADPA-IT-polypeptide (0.5 × 5cm) matrix of the urea of NaCl, the 4M of 7.3 the Tris/HCl that contains 50mM, 100mM, the selection of each polypeptide and in conjunction with being undertaken by described before method.Use the polypeptide solution that 1ml linear flow speed is 50cm/h.After injecting sample, the flow velocity of polypeptide solution is increased to 150cm/h.

Do not fix the cleaning of impurity component

Use the level pad of 5 times of chromatographic column volumes to clean not frozen composition.Be replaced by afterwards refolding damping fluid, the damping fluid of the sucrose of EDTA, the 0.25M of the Tris/HCl that contains 1M, 2mM that particularly pH value is 7.3, α-MTG (α-monothioglycerol) of 10M, this damping fluid volume is 6 times of chromatographic column volumes.

Refolding, shears and wash-out

After chaotropic environment change is cosmotropic environment, by stopping the mobile refolding that makes fusion polypeptide carry out 25h on chromatography resin of damping fluid.The sSPA-D that the activated self-proteolytic enzyme of tool connects C end shears.Use refolding damping fluid to carry out final wash-out and obtain natural sSPA-D.Use the NaOH of the 0.2M of the 10CV that flow velocity is 150cm/h to regenerate to matrix.

Embodiment 4:

6His-N ^prorefolding and the shearing of EDDIE-GFPmut3.1 in chromatographic column

The present embodiment has been described and has been used through 6His-N ^prothe process of natural GFPmut3.1 is prepared in the expression of EDDIE-GFPmut3.1 fusion polypeptide, and refolding and shearing are carried out on the affinity matrix that is called Actigel-polyKW.Chromatogram environment is with noted earlier identical.

Embodiment 5:

N ^prothe shearing of EDDIE-sSPA-D in chromatographic column

The present embodiment has been described use N ^pronatural sSPA-D is prepared in the expression of EDDIE-sSPA-D fusion polypeptide, and refolding and shearing are carried out on the affinity matrix that is called Actigel-polyKY.Chromatogram environment is with noted earlier identical.

Embodiment 6:

Use cation-exchange chromatography in chromatographic column, to carry out refolding

Crude product (Crude) N ^pro37-6His

It is in 7.0 the sodium radio-phosphate,P-32 solution that contains 8M urea, 50mM that ((1) MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDI RTTLRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFE TTKRIGRVTGSDGKLYHIYVEVDGEILLKLAKRGTPRTLKWTRNTTNCPLWVTSC-(168)) inclusion body extract (extracts) is suspended in pH value.The protein concn finally obtaining is 0.5mg/ml.2ml is loaded in HiTrap SP agarose FF chromatographic column (2.5 × 0.7cm i.d.; Chromatographic column volume is 1ml; GE Healthcare) upper, first use damping fluid same as described above to carry out balance with the linear flow speed of 50cm/h.Afterwards damping fluid is changed into pH value and is the damping fluid of α-monothioglycerol (MTG) of EDTA, 5% glycerol, the 10mM of 7 the sodium phosphate that includes 50mM, 2mM.At room temperature make protein carry out the refolding of 1h.Further use refolding damping fluid can be by the protein wash-out through refolding and shearing out.Use the sodium phosphate of the NaCl that contains 2M, 50mM that pH value is 7 can carry out regenerative operation.Analyze the refolding that can monitor fusion rotein (6His) by SDS-PAGE.

Claims (16)

1. a use includes required polypeptide and is connected to required polypeptide N end, the fusion polypeptide preparation with the polypeptide of self-proteolysis function has the method for the required polypeptide of allos of homology N end, the method comprises the following steps: a) use affinity chromatography system by fusion polypeptide with soluble, the form of oneself's proteolysis functionally inactive is fixed, b) refolding fusion polypeptide, to activate the self-proteolysis function of fusion polypeptide and required heterologous polypeptide sheared, c) the required heterologous polypeptide of wash-out subsequently, wherein said step is all carried out in an affinity chromatography system, the wherein said polypeptide with proteolysis function is the self-protease N of CSFV ^proor derivatives thereof, the self-protease N of described CSFV ^prothe aminoacid sequence of or derivatives thereof is as follows:

SEQ ID NO1：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFCEVTKRIGR VTGSDGKLYHIYVCVDGCILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168)，

SEQ ID NO2：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFEEVTKRIGR VTGSDGKLYHIYVEVDGEILLKLAKRGTPRTLKWIRNFTNCPLWVTSC-(168)，

SEQ ID NO3：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDEAQFEEVTKRIGR VTGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWIRNFTNCPLWVTSC-(168)，

SEQ ID NO 4：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGRGDIRTT LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV TGSDGKLYHIYVEVDGEILLKLAKRGTPRTLKWTRNTTNCPLWVTSC-(168)，

Or

SEQ ID NO 5：

(1)-MELNHFELLYKTSKQKPVGVEEPVYDTAGRPLFGNPSEVHPQSTLKLPHDRGEDDIETT LRDLPRKGDCRSGNHLGPVSGIYIKPGPVYYQDYTGPVYHRAPLEFFDETQFEETTKRIGRV TGSDGKLYHIYVEVDGEILLKQAKRGTPRTLKWTRNTTNCPLWVTSC-(168)。

2. method according to claim 1, wherein said fusion polypeptide is by being prepared from the recombinant expressed of inclusion body form in bacterial host cell, the encode expression vector transformed host cell of nucleic acid molecule of fusion polypeptide of use pass through to contain.

3. method according to claim 1 and 2, wherein said affinity chromatography system is selected from fixing metal ions chromatogram (IMAC), cation-exchange chromatography, anion-exchange chromatography, cellulose binding domain chromatogram or peptide affinity chromatography.

4. according to the method described in claim 3, wherein said affinity chromatography system is fixing metal ions chromatogram, and wherein said fusion polypeptide contains the affine mark of metal-chelating.

5. according to the method described in claim 4, wherein said metal-chelating is affine is labeled as poly Histidine.

6. according to the method described in claim 3, wherein said affinity chromatography system is cation-exchange chromatography, and wherein said fusion polypeptide contains polycation affinity labelling.

7. according to the method described in claim 6, wherein said polycation affinity labelling is selected from poly arginine or polylysine.

8. according to the method described in claim 3, wherein said affinity chromatography system is anion-exchange chromatography, and wherein said fusion polypeptide includes polyanion affinity labelling.

9. the method described according to Claim 8, wherein said polyanion is labeled as poly-asparagine.

10. according to the method described in claim 3, in wherein said peptide affinity chromatography system, using length is 5 to 12 oligopeptides aglucons amino acid length, that comprise tryptophan residue, in chaotropic environment, this aglucon optionally with in fusion polypeptide has the partial fixing of self-proteolysis function, when from chaotropic environment change to cosmotropic environment time, the part in this aglucon and fusion polypeptide with self-proteolysis function still keeps fixing.

11. according to the method described in claim 10, and the length of wherein said oligopeptides aglucon is 6 to 8 amino acid whose length.

12. according to the method described in claim 11, and the aminoacid sequence of wherein said oligopeptides aglucon is selected from:

SEQ ID NO6：VSIFEW，

SEQ ID NO7：AVSIEWY，

SEQ ID NO8：AVSFIWY，

SEQ ID NO9：VSFIWYK，

SEQ ID NO10：ASRFWYA，

SEQ ID NO11：AFYTWYA，

SEQ ID NO12：AFYRWYK，

SEQ ID NO13：AFYRWY，

SEQ ID NO14：AFYRWYA，

SEQ ID NO15：AVSIFEWY，

SEQ ID NO16：AVSRNWY，

SEQ ID NO17：ASRFWY，

SEQ ID NO18：AFYRWYAA，

SEQ ID NO19：AFYRWY，

SEQ ID NO20：ASRFWYAA，

SEQ ID NO21：AFYRWYAA，

SEQ ID NO22：AFYSWYAA。

13. according to the method described in claim 10, wherein said according to the N of the natural CSFV of SEQ ID NO5 ^proderivative, for be selected from oligopeptides aglucon as follows and combine

SEQ ID NO10：ASRFWYA，

SEQ ID NO11：AFYTWYA，

SEQ ID NO12：AFYRWYK，

SEQ ID NO13:AFYRWY and

SEQ ID NO14：AFYRWYA。

14. method according to claim 1, wherein the refolding step of fusion polypeptide is achieved chaotropic environment change to cosmotropic environment by changing damping fluid.

15. oligopeptides aglucon is the application in method described in any one in claim 10 to 13, the aminoacid sequence of wherein said oligopeptides aglucon is selected from:

SEQ ID NO6：VSIFEW，

SEQ ID NO7：AVSIEWY，

SEQ ID NO8：AVSFIWY，

SEQ ID NO9：VSFIWYK，

SEQ ID NO10：ASRFWYA，

SEQ ID NO11：AFYTWYA，

SEQ ID NO12：AFYRWYK，

SEQ ID NO13：AFYRWY，

SEQ ID NO14：AFYRWYA，

SEQ ID NO15：AVSIFEWY，

SEQ ID NO16：AVSRNWY，

SEQ ID NO17：ASRFWY，

SEQ ID NO18：AFYRWYAA，

SEQ ID NO19：AFYRWY，

SEQ ID NO20：ASRFWYAA，

SEQ ID NO21：AFYRWYAA，

SEQ ID NO22：AFYSWYAA。

The N of 16.CSFV ^proderivative application in method described in any one in claim 1 to 14, the N of described CSFV ^prothe aminoacid sequence of derivative be selected from SEQ ID NO2, SEQ ID NO3, SEQ ID NO4, SEQ ID NO5, SEQ ID NO32, SEQ ID NO50, SEQ ID NO51, SEQ ID NO52, SEQ ID NO53, SEQ ID NO54, SEQ ID NO55 or SEQ ID NO56.