CN101591660A - A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor - Google Patents

Abstract

The present invention relates to a kind ofly at intestinal bacteria secretion type expression recombinant human granulocyte-colony factor (rHG-CSF), this carrier is by adding DsbA signal peptide (SEQ ID NO:1) at the N of foreign protein end, and uses λ P _LPromotor can make up a kind of carrier of pericentral siphon solinocrine type express recombinant protein, and transformed into escherichia coli DH5 α competent cell is expressed in the colibacillus periplasm chamber by temperature-induced realization foreign protein.This method expression amount height, secernment efficiency height are applicable to secreting, expressing various exogenous genes efficiently and stably.

Description

A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor

Technical field

The present invention relates to a kind of colibacillus periplasm solinocrine type express recombinant human granulocyte-colony factor (G-CSF) method, more specifically, relate to a kind of the end and add DsbA signal peptide (MKKIWLALAGLVLAFSASA (SEQID NO:1)) by N at G-CSF, and the new expression vector that foreign protein is expressed in the colibacillus periplasm chamber; The explanation of this expression vector and application.

Technical background

Genetically engineered recombinant methionyl human G-CSF (rhG-CSF), be mainly used in the granulocytopenia after cancer patients's chemotherapy. be used for the oligoleukocythemia of leukemia, acquired immune deficiency syndrome (AIDS) of autologous bone marrow transplantation, some type and aplastic anemia etc. in addition, all show to have significant curative effect.Mostly by escherichia coli expression, though expression amount is high, form inclusion body at present, need just can obtain activated protein through the sex change renaturation, yield is very low, and N end methionine(Met) can not be removed.

Adopt prokaryotic expression system (mainly being coli expression system) to have the advantage of many uniquenesses., cell clear such as genetic background growth rapidly, fermentation period is short, be easy to screening and gene recombination operation, expression system is than horn of plenty etc.But also there are some shortcomings in prokaryotic expression system: can't glycosylation and be easy to form inclusion body etc. as foreign protein.The inclusion body that foreign protein forms must pass through sex change, fold renaturation again, just can obtain active protein product.The said process operation is loaded down with trivial details, annealing efficiency is low, need be optimized respectively for different foreign proteins.Therefore,, in application in practice, will increase work efficiency greatly so, save production cost if can adopt prokaryotic expression system to realize soluble-expression or the expression of pericentral siphon solinocrine in the born of the same parents of foreign protein.

People taked multiple mode to promote the soluble-expression of foreign protein.With gsh (GST) or sulphur oxygen cyclase protein amalgamation and expression, can play short solvable effect to the part foreign protein; Utilize protein disulfide to form the folding assisted effect of associated protein, cis-trans propyl isomerism enzyme and other molecular chaperoneses, design expression vector, can obviously promote the interior soluble-expression of born of the same parents of foreign protein.But, the soluble-expression mode also has apparent in view shortcoming in the born of the same parents: belong to the reductibility environment in the born of the same parents, be unfavorable for that foreign protein forms correct disulfide linkage bridge, and then influence the formation of correct structure picture, and need ultrasonication or pressure breaking cell walls could discharge soluble-expression component in the born of the same parents, be unfavorable for large scale culturing and purifying.Comparatively speaking, because the pericentral siphon chamber belongs to the oxidized form environment, help forming correct disulfide linkage bridge, and extract and do not need fracturing cell walls when component is expressed in the pericentral siphon chamber, can reduce the purifying cost significantly, be a kind of ideal phraseology so pericentral siphon solinocrine type is expressed.Usually foreign protein and appropriate signals peptide are merged, foreign protein is secreted in the pericentral siphon chamber by the guiding function of signal peptide.The copy number of control expression vector is selected suitable promotor and substratum, adopts suitable abduction delivering mode and culture condition (culture temperature, induce opportunity and induction time), can improve the efficient of pericentral siphon solinocrine type expression.

Experiment showed, the G-CSF that is connected to behind the signal peptide, realize that not only pericentral siphon solinocrine type expresses, the G-CSF of expression need not renaturation obtain activated product, and correct through the order-checking protein structure, N-holds no unnecessary amino acid.The principle of secreting, expressing is: purpose G-CSF encoding sequence is fused to after the signal coding sequence, expression product is when secreting to born of the same parents' pericentral siphon (periPlasmic space) by the Bacillus coli cells inner membrance, signal peptide can be by correct identification of signal peptidase and excision.Foreign protein after the secretion does not contain N end methionine(Met), and space folding (comprise and form disulfide linkage) one-tenth has had the activated protein of native conformation, need not follow-up renaturation process, thereby simplified production technique. in addition, the foreign protein that is accumulated in born of the same parents' pericentral siphon has been avoided the degraded of some proteolytic enzyme in the cell, thereby more stable.

Summary of the invention

Purpose of the present invention just provides the method that a kind of prokaryotic secretion is expressed G-CSF, and the versatility of this method is good, expression amount is high, secernment efficiency is high, is applicable to secreting, expressing various exogenous genes efficiently and stably, as G-CSF, IL-2 etc.

The present invention relates to recombinant DNA technology, relate more specifically to the construction process that a kind of new prokaryotic secretion is expressed the G-CSF plasmid.The invention still further relates to prokaryotic secretion and express expression and the extracting method of G-CSF.

Description of drawings

The structure iron of Fig. 1 pDsbA-GCSF.

The structure iron of Fig. 2 pBV-DsbA-GCSF.

Fig. 3 is the chamber protein electrophoresis between week

When expressing GCSF, gene is inserted into the downstream of DsbA sequence, this is so that during expressing gene, expressed peptide chain is by DsbA Signal peptide is directed in the pericentral siphon chamber and is folded to form activated protein. Selected in the method the strong terminator sequence of rrnB, and also had steady Tailor-made using. As for elements such as origin of replication ori and marker gene (such as resistant gene), without any particular limitation. Such as, use Can select tetracycline resistance gene, neomycin resistance gene or ammonia joint penicillin resistance gene etc. in the resistant gene of screening.

In another aspect of the present invention, provide a kind of host of containing general pericentral siphon solinocrine type expression vector pDsbA thin Born of the same parents, commonly used is bacillus coli DH 5 alpha, is coli strain W3110 best.

Carrier provided by the present invention has following characteristics:

1. the short folded signal peptide DsbA of coexpression effectively promotes correct fold of GCSF in the pericentral siphon chamber, improves the activated protein expression level;

2. when adopting this carrier to carry out the expression of pericentral siphon solinocrine type, do not need broken bacteria cell wall, directly extract pericentral siphon chamber component and promptly can gather in the crops target protein.

3. expressed proteins all is the activated protein that function is arranged by this way, does not need complex steps and extremely low sex change and the renaturation process of efficient, only needs comparatively simple purification step just can obtain a large amount of foreign proteins;

4. this expression vector is expressive host with intestinal bacteria, but bulk fermentation production, production technique is simple, and operation is with low cost easily.

Embodiment

In an example of the present invention, in order to obtain to make up several members of secreted expression carrier, at first according to known λ P _LThe sequence of promotor, the synthetic PCR primer of use, increasing from escherichia coli vector pBLV carrier with the method for PCR obtains λ P _LPromotor.Sequences Design according to DsbA is extended primer, with existing GCSF is template, with the N end of pcr amplification to GCSF, DsbA-GCSF fragment after pBR322 fragment after two ends design restriction enzyme site is cut suitable enzyme then, promoter sequence and suitable enzyme are cut links together, and has just formed a kind of new GCSF secretion expression carrier pDsbA-GCSF.Also DsbA-GCSF is building up to simultaneously the pBv220 carrier and forms pBV-DsbA.Two kinds of plasmids are transformed DH5 α respectively.

For the transformed host cells that filters out, can under the usual culture condition that uses in this area, cultivate to express GCSF, wherein very important to the control of the OD value before inducing, suitable induced concentration can make the GCSF expression rate higher.Cultivate the OD value when the 0.8-1.0 when 30 ℃, elevated temperature to 42 ℃ is induced and was collected thalline in 6 hours rapidly, and GCSF has preferably and expresses.

Embodiment 1

The sequence of λ PL promotor is synthesized and is used the PCR primer, clones from escherichia coli vector pBLV with the method for PCR to obtain, and be that template is carried out pcr amplification with it.Used upstream primer is: 5 ' GTGAATTCAGATCTCTCACCTACCAAAC3 ' (containing EcoRI), downstream primer is: 5 ' GTCATATGACTAGTCCTCCTTAATTTTTAACCAATG 3 ' (containing the Spel site).The PCR product is behind the EcoRI/Spel double digestion, and low melting point glue reclaims standby.

Embodiment 2

People GCSF gene is that my company is existing, design primer PCR from former preservation plasmid obtains, primer is extended in two of upstream designs, primer 1:5 '-GGT TTA GTT TTA GCG TTT AGC GCA TCG GCGACACCATTAGGCCCTGCCAGC-3 ', primer 2: 5 '-GAGGAATTCATG AAA AAG ATT TGG CTGGCG CTG GCT GGT TTA GTT TTA GCG TTT AGC-3 ' downstream primer, 5 '-GCGGGATCCTCACGGCTGGGCAAG-3 ', the fragment that amplifies is about 600bp.Second GCA of 5 ' end links to each other with last amino acid whose coding DNA of DsbA signal peptide, thereby first amino acid of coding people GCSF maturation protein is (Thr), therefore signal peptide excision back excretory GCSF N-terminal does not contain Met, and consistent with natural GCSF.The aminoacid sequence of signal peptide is: Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser Ala SerAla

Embodiment 3

The structure of pBV-DsbA-GCSF plasmid

In this embodiment, make up the plasmid that contains DsbA-GCSF sequence and rmB terminator sequence. that the rmB sequence is provided is plasmid pBv220, and this is a kind of known expression plasmid.The heterologous protein sequence that will express in this plasmid is placed in the downstream of PRPL promotor, and this promotor can make and be positioned at its downstream albumen sequence table and reach when thermal induction.Cut among the plasmid pBV220 with BamHI/SaLI is two, form plasmid pBV-DsbA-GCSF.The structure of PBV-DsbA-GCSF plasmid as shown in Figure 2, what be close to DsbA-GCSF sequence downstream is exactly the rrnB terminator sequence.

Embodiment 4

The structure of expression vector pDsbA with EcoRI/SpeI double digestion plasmid pBR322, is isolated big fragment as shown in Figure 4 then.Plasmid pBV-DsbA-GCSF with obtaining among the EcoRI/SpeI double digestion embodiment 3 separates small segment then.Above-mentioned 2 kinds of isolating fragments are mixed by a certain percentage, connect with ligase enzyme and spend the night, thereby be built into plasmid pDsbA-GCSF.Then this recombinant expression vector is changeed people intestinal bacteria W3110, set up the engineering bacteria PDsbA-hGCSF/W3110 of people GCSF secretion type expression.

For the transformed host cells that filters out, can under the usual culture condition that uses in this area, cultivate to express GCSF, wherein very important to the control of the OD value before inducing, suitable induced concentration can make the GCSF expression rate higher.Cultivate the OD value when the 0.8-1.0 when 30 ℃, elevated temperature to 42 ℃ is induced and was collected thalline in 6 hours rapidly, and GCSF has preferably and express (account between week chamber proteic more than 40%) as Fig. 3.

The extraction of GCSF and purifying

GCSF slightly carries: slightly carrying damping fluid is 10mMTris-HCL (pH7.4) (20% sucrose), and every mL adds 33ul0.5mMEDTA.Thick extracting method: take out thalline from-70 ℃, add the above-mentioned damping fluid of people 10ml by the wet bacterium of every gram, the suspension bacterium, be placed on 4 ℃ of vibration 1h, suspension is through 4 ℃, 12000g, 20min is centrifugal, and supernatant is collected in the back, and supernatant is behind the positive press filtration of 5um filter membrane, be the GCSF extracting solution. cross the cation-exchange chromatography post, pillar with sample on the 10ml/min flow velocity, is used the GCSF crude extract instead the 20ml/min flow velocity after the abundant balance of buffer A (20mMNaAC-HAC pH5.0), with 20% buffer B (IMNaCI, 20mMNaAC-HACpH5.0) wash post to the 280nm uv-absorbing to baseline, wash post with 30% buffer B again, collect the eluted protein peak.

Hydrophobic chromatography: filler is the Phemyl-sepharose HP of Pharmacia company.Post is through level pad A (1.5M (NH4) 2S04,20mM NaAC-HACpH5.0) abundant balance. add people's solid (NH4) 2S04 in the component that ion exchange chromatography obtains, making its final concentration is 1.5M, the centrifugal post precipitation that goes is with sample on the 5ml/min flow velocity, use the 10ml/min flow velocity instead, wash post to the 280nm uv-absorbing to baseline with 40% buffer B (20mM NaAC-HAC pH5.0), remake the 40-60%B linear gradient elution, collect GCSF activated protein peak.

Ultrafiltration and concentration:, on the Minitan ultra-fine filter, use PTGC filter membrane (MW10000) ultrafiltration and concentration to 90-100ml the GCSF active constituent that hydrophobic chromatography obtains.Gel-filtration: filler is the Sephacryl S-100 of Pharmacia company, and column volume is 5cm * 115cm.After the earlier abundant balance of pillar, with sample chromatography on the ultrafiltration and concentration sample, flow velocity is 60ml/h, collects GCSF activated protein peak.The evaluation of GCSF behind the purifying: to the GCSF behind the aforesaid method purifying, carry out the HPLC purity testing with ordinary method, the result records its purity greater than 98%.

In this embodiment, GCSF all is secreted into the Bacillus coli cells pericentral siphon more than 95% when the optimum condition bottom fermentation finishes, expression amount accounts for 40% of born of the same parents' periplasm protein total amount, the output of GCSF is 350-500mg in the 1L fermented liquid, and this is than so far for to exceeding nearly 5 times with the GCSF productive rate of inclusion body formal representation.In addition needn't renaturation, so technology is easy, and the activity of the GCSF that obtains is very high.

Claims (3)

1. a pericentral siphon solinocrine type is expressed the method for this stimulating factor of express recombinant human granulocyte colony, and its structure as shown in Figure 1.The N end that it is characterized in that protein gene contains signal peptide.

2. the expression method of claim 1, wherein said signal peptide is DsbA signal peptide SEQ ID NO:1, uses λ P _LPromotor utilizes the temperature adjusting induction exogenous gene to express.

3. the purposes of claim 1,2 described methods is characterized in that expressing in the cell pericentral siphon by signal peptide guiding recombinant human granulocyte-colony factor rHG-CSF, helps forming being dissolved with activated protein, need not break bacterium, directly extracts albumen from the pericentral siphon chamber.

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